Autophagy Of Endothelial Progenitor Cells Research Articles

Upregulated miR-206 Aggravates Deep Vein Thrombosis by Regulating GJA1-Mediated Autophagy of Endothelial Progenitor Cells.

Background Deep vein thrombosis (DVT) is the third most prevalent vascular disease worldwide. MicroRNAs (miRNAs) play regulatory roles in functions of endothelial progenitor cells (EPCs), which is becoming a promising therapeutic choice for thrombus resolution. Nevertheless, the role of miR-206 in EPCs is unclear. Methods EPCs were isolated from the peripheral blood of patients with DVT. In DVT mouse models, DVT was induced by stenosis of the inferior vena cava (IVC). The levels of miR-206 and gap junction protein alpha 1 (GJA1) in EPCs and vascular tissues of DVT mice were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The proliferation, migration, apoptosis, and angiogenesis were tested by cell counting kit-8 (CCK-8) assay, Transwell assay, flow cytometry analysis, and in vitro tube formation assay. The levels of autophagy-related proteins as well as the level of GJA1 in EPCs and vascular tissues were evaluated by western blotting. DVT formation in vivo was observed through hematoxylin-eosin (HE) staining. The expression of thrombus resolution markers, CD34 molecule (CD34) and matrix metallopeptidase 2 (MMP2), in the thrombi was measured by immunofluorescence staining. Results miR-206 overexpression inhibited proliferation, migration, and angiogenesis and promoted apoptosis of EPCs, while miR-206 knockdown exerted an opposite effect on EPC phenotypes. Downregulation of GJA1, the target of miR-206, abolished the influence of miR-206 on EPC phenotypes. Furthermore, silencing of miR-206 suppressed the autophagy of EPCs via upregulating GJA1. miR-206 knockdown repressed thrombus formation, enhanced the homing ability of EPCs to the thrombosis site, and facilitated thrombus resolution in DVT mouse models. Additionally, miR-206 was upregulated while GJA1 was downregulated in vascular tissues of DVT mice. miR-206 knockdown elevated GJA1 expression in vascular tissues of DVT mice. The expression of miR-206 was negatively correlated with that of GJA1 in DVT mice. Conclusion miR-206 knockdown upregulates GJA1 to inhibit autophagy of EPCs and then promote EPC proliferation, migration, and angiogenesis, thereby enhancing EPC homing to thrombi and facilitating thrombus resolution.

THRIL mediates endothelial progenitor cells autophagy via AKT pathway and FUS

BackgroundThis study focused on the roles of lncRNA THRIL in coronary atherosclerotic heart disease (CAD) through regulating AKT signaling pathway and directly interacting with FUS.MethodsQRT-PCR was conducted to detect the expression of THRIL in CAD blood samples and endothelial progenitor cells (EPCs). Cell autophagy of EPCs was examined through Cyto-ID Autophagy Detection Kit. CCK-8 assay and flow cytometry were carried out to assess cell viability and apoptosis under various interference conditions. Western blotting was conducted to detect the expression of interest proteins. The expression levels of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were measured by qRT-PCR. The direct interactions between HCG18 and FUS was confirmed through RNA electrophoretic mobility shift assay (RNA EMSA) and RNA immunoprecipitation (RIP) assay.ResultsTHRIL was upregulated in CAD blood samples and EPCs. Knockdown of THRIL in EPCs promoted cell viability, inhibited cell autophagy and further suppressed the development of CAD. Over-expression of THRIL induced inactivation of AKT pathway, while knockdown of THRIL played reversed effects. THRIL directly interacted with FUS protein and knockdown of FUS reversed the over-expressing effect of THRIL on cell proliferation, autophagy and the status of AKT pathway.ConclusionTHRIL inhibits the proliferation and mediates autophagy of endothelial progenitor cells via AKT pathway and FUS.

Fasting Therapy Contributes to the Improvement of Endothelial Function and Decline in Vascular Injury‐Related Markers in Overweight and Obese Individuals via Activating Autophagy of Endothelial Progenitor Cells

Background High body mass index- (BMI-) related vascular injury contributes to the pathogenesis of the atherosclerotic cardiovascular disease (ASCVD). Rigorous calorie restriction is one of the major lifestyle interventions to reduce vascular risk in overweight or obese individuals. However, the effects of fasting therapy (FT) on vascular function and the mechanism are still unclear. This study was aimed to investigate the impacts of FT on endothelial function, arterial stiffness, and circulating arterial damage parameters in overweight and obese individuals and possible mechanism. Methods Overweight and obese individuals participated in FT intervention (7-day very low calorie diet). Arterial function including brachial arterial flow-mediated dilation (FMD), brachial-ankle pulse wave velocity (baPWV), vascular injury-related markers including trimethylamine N-oxide (TMAO), and leptin and endothelial microparticles (EMPs) were assessed. Endothelial progenitor cells (EPCs) of these participants were isolated and cultured to investigate EPCs function. mRFP-GFP-LC3 confocal microscopy scanning and western blot were carried out to determine autophagy. Results After FT, body weight and BMI significantly decreased (81.76 ± 12.04 vs. 77.51 ± 12.06 kg, P < 0.01; 29.93 ± 2.82 vs. 28.47 ± 2.83 kg/m2, P < 0.01). FT remarkably improved FMD (5.26 ± 1.34 vs. 6.25 ± 1.60%, P=0.01) while baPWV kept unchanged. TMAO and leptin levels decreased (3.96 ± 1.85 vs. 2.73 ± 1.33 μmol/L, P=0.044; 6814 ± 2639 vs. 3563 ± 2668 μmol/L, P < 0.01). EMPs showed a decreased tendency. EPCs function was significantly improved, autophagy fluorescence intensity was enhanced, and the level of Beclin1, Atg5, LC3 II/I also increased after starvation in vitro, and the effects were blocked by autophagy inhibitor. Conclusions Our present study demonstrated for the first time that FT markedly improves endothelial function and reduces the levels of arterial injury markers through improving EPCs function via activating autophagy. These findings provide a novel insight into FT as a lifestyle intervention strategy to promote the maintenance of vascular homeostasis in overweight or obese individuals. The trial was registered with ChiCTR1900024290.

Clarification of the Role of miR-9 in the Angiogenesis, Migration, and Autophagy of Endothelial Progenitor Cells Through RNA Sequence Analysis

We have previously reported that miR-9 promotes the homing, proliferation, and angiogenesis of endothelial progenitor cells (EPCs) by targeting transient receptor potential melastatin 7 via the AKT autophagy pathway. In this way, miR-9 promotes thrombolysis and recanalization following deep vein thrombosis (DVT). However, the influence of miR-9 on messenger RNA (mRNA) expression profiles of EPCs remains unclear. The current study comprises a comprehensive exploration of the mechanisms underlying the miR-9-regulated angiogenesis of EPCs and highlights potential treatment strategies for DVT. We performed RNA sequence analysis, which revealed that 4068 mRNAs were differentially expressed between EPCs overexpressing miR-9 and the negative control group, of which 1894 were upregulated and 2174 were downregulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that these mRNAs were mainly involved in regulating cell proliferation/migration processes/pathways and the autophagy pathway, both of which represent potential EPC-based treatment strategies for DVT. Reverse transcriptase quantitative polymerase chain reaction confirmed the changes in mRNA expression related to EPC angiogenesis, migration, and autophagy. We also demonstrate that miR-9 promotes EPC migration and angiogenesis by regulating FGF5 directly or indirectly. In summary, miR-9 enhances the expression of VEGFA, FGF5, FGF12, MMP2, MMP7, MMP10, MMP11, MMP24, and ATG7, which influences EPC migration, angiogenesis, and autophagy. We provide a comprehensive evaluation of the miR-9-regulated mRNA expression in EPCs and highlight potential targets for the development of new therapeutic interventions for DVT.

Inhibition of Galectin-3 Alleviates Cigarette Smoke Extract-Induced Autophagy and Dysfunction in Endothelial Progenitor Cells.

Endothelial progenitor cells (EPCs) have the potential to repair damaged blood vessels and promote angiogenesis. Smoking, an important risk factor for cardiovascular diseases, is associated with impaired functions of EPCs. However, the underlying mechanisms remain unclear. The aim of the study was to investigate the effects of cigarette smoke extract (CSE) on autophagy and dysfunction of EPCs and the involvement of galectin-3 in its effects. EPCs were treated with 8% CSE for 24 h (without affecting cell viability). EPC functions were assessed by tube formation and migration capacity and intracellular ROS and eNOS expression. Autophagy was assessed by autophagic protein expression by Western blotting and immunofluorescence microscopy and autophagosome accumulation by transmission electron microscopy. Galectin-3 expression was measured by real-time PCR, Western blotting, and immunofluorescence microscopy, while phospho-AMPK and phospho-mTOR were measured by Western blotting. EPCs were transfected by shRNA-Gal-3 or shRNA-NC before treatment with CSE to examine the effects of galectin-3 on CSE-induced autophagy and dysfunction of EPCs. CSE-treated EPCs showed decreased tube formation and migration ability and eNOS expression but increased oxidative stress. CSE also induced autophagy which was characterized by a decrease in p62 protein, an increase in LC3B-II/I ratio, and accumulation of autophagosomes. CSE upregulated galectin-3 expression on EPCs. Inhibition of galectin-3 abrogated CSE-induced autophagy and dysfunction of EPCs. CSE activated phospho-AMPK and inhibited phospho-mTOR, and inhibition of galectin-3 abolished CSE's effect on activating phospho-AMPK and inhibiting phospho-mTOR. In conclusion, our results suggest that galectin-3 mediates CSE-induced EPC autophagy and dysfunction, likely via the AMPK/mTOR signaling pathway.

MeCP2 inhibits cell functionality through FoxO3a and autophagy in endothelial progenitor cells.

Objectives: Autophagy is an evolutionarily conserved intracellular degradation mechanism in which cell constituents are phagocytosed to maintain cellular homeostasis. Forkhead box O 3a (FoxO3a) promotes autophagy to protect cells from environmental stress. Methylated CpG binding protein 2 (MeCP2) is a nuclear protein that binds DNA and represses transcription. However, the mechanism and interplay between FoxO3a and MeCP2 underlying endothelial progenitor cell (EPC) function are not fully understood.Results: In EPCs, MeCP2 overexpression attenuated autophagy and cell functionality, which were reversed by the autophagy activator rapamycin or co-transfection with FoxO3a. FoxO3a promoted cell function, which was reversed by the autophagy inhibitor chloroquine. Following MeCP2 overexpression, MeCP2 was found enriched on the FoxO3a promoter, resulting in promoter hypermethylation and enhanced H3K9 histone modification in nucleosomes of the FoxO3a promoter.Conclusions: MeCP2 attenuated cell functionality via DNA hypermethylation and histone modification of the FoxO3a promoter to inhibit FoxO3a transcription and autophagy.Materials and Methods: EPCs were isolated from human umbilical cord blood and treated with adenoviral vectors containing interference sequences. The effects and mechanism of MeCP2 and FoxO3a were analyzed by utilizing western blotting, cell counting kit-8, transwell plates, Matrigel, matrix adhesion, transmission electron microscopy, and chromatin immunoprecipitation.

MALAT1/miR-15b-5p/MAPK1 mediates endothelial progenitor cells autophagy and affects coronary atherosclerotic heart disease via mTOR signaling pathway.

Objective: Present study focused on the influence of lncRNA MALAT1 on coronary atherosclerotic heart disease (CAD) by regulating miR-15b-5p/MAPK1 and mTOR signaling pathway.Method: Differentially expressed genes and activated pathway were investigated through bioinformatics analysis. QRT-PCR was conducted to verify expression of MALAT1, miR-15b-5p and MAPK1 in CAD blood samples and endothelial progenitor cells (EPCs). In addition, the interactions among MALAT1, miR-15b-5p and MAPK1 were revealed by Luciferase reporter assay. Cell autophagy of EPCs was examined by Cyto-ID Autophagy Detection Kit and transmission electron microscope. MTT assay and flow cytometry were carried out to assess cell viability and apoptosis in different interference conditions. Western blot was performed to testify the expression of pERK1/2 (MAPK1), phosphorylated mTOR, ATG1 and LC3-II. Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were detected by qRT-PCR. Finally, the effect of lncRNA MALAT1 on cell autophagy and atherogenesis was tested in vivo.Results: MALAT1 was overexpressed in CAD blood samples and EPCs. Knockdown of MALAT1 and MAPK1 promoted cell viability, autophagy and further suppressed the development of CAD. AntagoMALAT1 protects mice against atherosclerosis.Conclusion: LncRNA MALAT1 inhibited EPCs autophagy and increased cell viability while repressed apoptosis of CAD via activating mTOR signaling pathway.

The Role of Autophagy in the Differentiation of EPCs Induced by Shear Stress

Endothelial progenitor cells (EPCs) play an important role in postnatal angiogenesis and neovascularization. Previous studies have revealed shear stress could accelerate EPC proliferation, differentiation, migration and so on, which contribute to postnatal angiogenesis and neovascularization. Moreover, some studies indicate that autophagy actively participates angiogenesis by affecting EPC migration and differentiation. Here, we try to elucidate the possible roles of autophagy of EPC differentiation induced by shear stress. Methods and Results：EPCs were exposed to shear stress (12 dyne/cm2). And then the expression of autophagy markers, such as LC3Ⅱ/Ⅰ,P62andATG5, were analyzed using Western blot. The results have shown that in EPCs, shear stress triggered an increase in LC3Ⅱ/Ⅰ and ATG5 at 10 min, which was then followed by a decrease. In contrast, shear stress caused a decrease in P62 at 10 min, which was then followed by an increase. Furthermore, immunostaining revealed that the unsheared cells showed only weak LCⅡ staining. However, shear stress increased LCⅡ staining. Bafromycin experiment confirmed that the increase of autophagy caused by shear stress was due to an increase in the formation of autophagy rather than a decrease in the degradation of autophagosomes. To examine the role of autophagy in the shear stress-induced EPC differentiation, we pretreated late EPCs with 3-MA, an inhibitor of autophagy, before the application of shear stress. Through real time RT-PCR and FACS analyses, we observed that the pretreatment of EPCs with 3-MA significantly inhibited the shear stress induced up-regulation of vWF and CD31. In the mean time, treatment of EPCs with LY294002 (a small molecule inhibitor of PI3K) or KLF2 siRNA inhibited the shear stress-induced EPC autophagy and differentiation. Conclusion：Autophagy is involved in the shear stress-upregulated expression of endothelial markers vWF and CD31 in EPCs. Moreover, this increase was observed to be mediated by PI3K and KLF2. Although further studies are needed to confirm the relationship between these mechanosensitive molecules, the present results may provide new insights into the relationship between EPC autophagy and differentiation induced by shear stress.

Monotropein promotes angiogenesis and inhibits oxidative stress-induced autophagy in endothelial progenitor cells to accelerate wound healing.

Attenuating oxidative stress‐induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress‐induced mitochondrial dysfunction and stimulate bone marrow‐derived EPC (BM‐EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM‐EPCs and prevented tert‐butyl hydroperoxide (TBHP)‐induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated via mTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM‐EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury‐related wounds.

Sitagliptin-mediated preservation of endothelial progenitor cell function via augmenting autophagy enhances ischaemic angiogenesis in diabetes.

Recently, the dipeptidyl peptidase‐4 (DPP‐4) inhibitor sitagliptin, a major anti‐hyperglycaemic agent, has received substantial attention as a therapeutic target for cardiovascular diseases via enhancing the number of circulating endothelial progenitor cells (EPCs). However, the direct effects of sitagliptin on EPC function remain elusive. In this study, we evaluated the proangiogenic effects of sitagliptin on a diabetic hind limb ischaemia (HLI) model in vivo and on EPC culture in vitro. Treatment of db/db mice with sitagliptin (Januvia) after HLI surgery efficiently enhanced ischaemic angiogenesis and blood perfusion, which was accompanied by significant increases in circulating EPC numbers. EPCs derived from the bone marrow of normal mice were treated with high glucose to mimic diabetic hyperglycaemia. We found that high glucose treatment induced EPC apoptosis and tube formation impairment, which were significantly prevented by sitagliptin pretreatment. A mechanistic study found that high glucose treatment of EPCs induced dramatic increases in oxidative stress and apoptosis; pretreatment of EPCs with sitagliptin significantly attenuated high glucose‐induced apoptosis, tube formation impairment and oxidative stress. Furthermore, we found that sitagliptin restored the basal autophagy of EPCs that was impaired by high glucose via activating the AMP‐activated protein kinase/unc‐51‐like autophagy activating kinase 1 signalling pathway, although an autophagy inhibitor abolished the protective effects of sitagliptin on EPCs. Altogether, the results indicate that sitagliptin‐induced preservation of EPC angiogenic function results in an improvement of diabetic ischaemia angiogenesis and blood perfusion, which are most likely mediated by sitagliptin‐induced prevention of EPC apoptosis via augmenting autophagy.

Protective effect and mechanism of lycopene on endothelial progenitor cells (EPCs) from type 2 diabetes mellitus rats

Endothelial progenitor cells (EPCs), widely existing in bone marrow and peripheral blood, are involved in the repair of injured vascular endothelium and angiogenesis which are important to diabetic mellitus (DM) patients with vascular complications. The number and the function of EPCs are related to the advanced glycation end products (AGEs) generated in DM patients. Lycopene (Lyc) is an identified natural antioxidant that protects EPCs under the microenvironment of AGEs from damage. However, the underlying mechanism remains unclear. To investigate the effect of Lyc on EPCs, we isolated EPCs from DM rat bone marrow and determined cell proliferation, cell cycle,apoptosis and autophagy of EPCs. The present study showed that 10μg/mL Lyc improved cell proliferation and had low cytotoxicity in the presence of AGEs. In addition, Lyc rescued S phase of the cell cycle arrest, reduced apoptosis rate and decreased autophagic reaction including ROS and mitochondrial membrane potential (MMP) of EPCs. Moreover, Lyc combined use of autophagy inhibitors, 3-MA, had better protective effects. Taken together, our data suggests that Lyc promotes EPCs survival and protect EPCs from apoptosis and oxidative autophagy induced by AGEs, further remaining the number and function of EPCs. This study provides new insights into Lyc protective mechanism of AGEs-induced oxidative autophagy in EPCs from DM patients and offers a new therapy for DM vascular complications.

MicroRNA-195 regulates proliferation, migration, angiogenesis and autophagy of endothelial progenitor cells by targeting GABARAPL1.

Deep vein thrombosis (DVT) is a common type of venous thrombosis. Successful resolution of DVT-related thrombi is important in the treatment of DVT. Endothelial progenitor cells (EPCs) have emerged as a promising therapeutic choice for DVT-related thrombus resolution; however, the clinical application of EPCs faces many challenges. In the present study, the expression of miR-582, miR-195 and miR-532 under hypoxic or normoxic conditions was measured using quantitative real-time PCR analysis (qRT-PCR) and the results showed that the increased fold of miR-195 was highest in human EPCs (hEPCs) under hypoxic conditions. Then the role and regulating mechanism of miR-195 in improving the function of EPCs was investigated. To investigate the effect of miR-195 inhibition on the autophagy of hEPCs, the expression of the autophagy-related genes LC3B and beclin1 was examined using western blotting, and the formation of autophagosomes was observed using TEM. The results indicated that the inhibition of miR-195 expression could promote autophagy of hEPCs. In addition, we investigated the role of miR-195 on the proliferation, migration and angiogenesis of hEPCs under hypoxia. The results revealed that miR-195 inhibition promotes cell proliferation, migration and angiogenesis of hEPCs under hypoxia. Furthermore, GABA type A receptor associated protein like 1 (GABARAPL1) was identified as a directed target of miR-195 and GABARAPL1 silencing could decrease the effect of miR-195 knockdown on cell proliferation, migration, angiogenesis and autophagy of hEPCs under hypoxia. Together, these results indicate that miR-195 regulates cell proliferation, migration, angiogenesis and autophagy of hEPCs by targeting GABARAPL1.

Transcription factor E2-2 inhibits the proliferation of endothelial progenitor cells by suppressing autophagy.

Endothelial progenitor cells (EPCs) play a key role in repairing the injured vascular endothelium by differentiating into mature endothelial cells (ECs) or secreting cytokines in a paracrine manner to promote proliferation of existing ECs. However, the mechanisms underlying the proliferation of EPCs were not fully understood. In order to investigate the mechanisms of EPC proliferation, we isolated EPCs from mononuclear cells of mouse spleens. By manipulating E2-2 expression in vitro, we observed that E2-2 negatively regulated the proliferation of EPCs. Moreover, we noted that E2-2 negatively regulated the autophagy of EPCs by studying the expression of LC3II and p62. We also demonstrated that an autophagy inhibitor chloroquine (CQ) decreased the proliferation of EPCs in a concentration-dependent manner. Interestingly, CQ reversed the increase in cell proliferation and autophagy in the E2-2 knockdown group. Furthermore, we detected the expression of autophagy-related protein ATG7 in EPCs which had been transfected with small interfering (siRNA)-E2-2 and siRNA-autophagy related 7 (ATG7) or were untransfected. Our study revealed that E2-2 regulated EPC autophagy via mediating ATG7 expression. We conclude that E2-2 inhibited EPC proliferation via suppressing their autophagy, and E2-2 regulated EPC autophagy by mediating the expression of ATG7.

Adenosine accelerates the healing of diabetic ischemic ulcers by improving autophagy of endothelial progenitor cells grown on a biomaterial.

Endothelial progenitor cells (EPCs) seeded on biomaterials can effectively promote diabetic ischemic wound healing. However, the function of transplanted EPCs is negatively affected by a high-glucose and ischemic microenvironment. Our experiments showed that EPC autophagy was inhibited and mitochondrial membrane potential (MMP) was increased in diabetic patients, while adenosine treatment decreased the energy requirements and increased the autophagy levels of EPCs. In animal experiments, we transplanted a biomaterial seeded with EPCs onto the surface of diabetic wounds and found that adenosine-stimulated EPCs effectively promoted wound healing. Increased microvascular genesis and survival of the transplanted cells were also observed in the adenosine-stimulated groups. Interestingly, our study showed that adenosine increased the autophagy of the transplanted EPCs seeded onto the biomaterial and maintained EPC survival at 48 and 96 hours. Moreover, we observed that adenosine induced EPC differentiation through increasing the level of autophagy. In conclusion, our study indicated that adenosine-stimulated EPCs seeded onto a biomaterial significantly improved wound healing in diabetic mice; mechanistically, adenosine might maintain EPC survival and differentiation by increasing high glucose-inhibited EPC autophagy and maintaining cellular energy metabolism.

Icariin promotes angiogenic differentiation and prevents oxidative stress-induced autophagy in endothelial progenitor cells.

Reduced tissue levels of endothelial progenitor cells (EPCs) and functional impairment of endothelium are frequently observed in patients with diabetes and cardiovascular disease. The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Hence attention has increasingly been paid to enhance mobilization and differentiation of EPCs for therapeutic purposes. The aim of this study was to investigate whether Icariin, a natural bioactive component known from traditional Chinese Medicine, can induce angiogenic differentiation and inhibit oxidative stress-induced cell dysfunction in bone marrow-derived EPCs (BM-EPCs), and, if so, through what mechanisms. We observed that treatment of BM-EPCs with Icariin significantly promoted cell migration and capillary tube formation, substantially abrogated hydrogen peroxide (H2 O2 )-induced apoptotic and autophagic programmed cell death that was linked to the reduced intracellular reactive oxygen species levels and restored mitochondrial membrane potential. Icariin downregulated endothelial nitric oxide synthase 3, as well as nicotinamide-adenine dinucleotide phosphate-oxidase expression upon H2 O2 induction. These antiapoptotic and antiautophagic effects of Icariin are possibly mediated by restoring the loss of mammalian target of rapamycin /p70S6K/4EBP1 phosphorylation as well as attenuation of ATF2 and ERK1/2 protein levels after H2 O2 treatment. In summary, favorable modulation of the angiogenesis and redox states in BM-EPCs make Icariin a promising proangiogenic agent both enhancing vasculogenesis and protecting against endothelial dysfunction.

MicroRNA-130a regulates autophagy of endothelial progenitor cells through Runx3.

Dysfunction of endothelial progenitor cells (EPC) contribute to diabetic vascular disease. MicroRNAs (miRNAs) are key regulators of diverse cellular processes, including angiogenesis. We recently reported that downregulated miR-130a in patients with Type 2 diabetes mellitus (DM) results in EPC dysfunction, including increased apoptosis, likely via its target runt-related transcription factor 3 (Runx3). However, whether miR-130a affects the autophagy of EPC is unknown. The aim of the present study was to explore the effects of miR-130a on the autophagy and cell death of EPC, as well as their expression of Beclin 1 (BECN1; an initiator of autophagosome formation) and the anti-apoptotic protein Bcl2 (which binds to and inactivates BECN1), and the role of Runx3 in mediating these effects. The EPC were cultured from peripheral blood mononuclear cells of diabetic patients and non-diabetic controls. Cells were transfected with an miR-130a inhibitor, or mimic-miR-130a or mimic-miR-130a plus lentiviral vector expressing Runx3 to manipulate miR-130a and/or Runx3 levels. The number of autophagosomes was counted under transmission electron microscopy and cell death was examined by flow cytometry. The mRNA expression of Beclin1 was measured by real-time polymerase chain reaction and the protein expression of Beclin1 and Bcl2 was determined by western blotting. Both the number of autophagosomes and Beclin1 expression were increased in EPC from patients with DM. Inhibition of miR-130a increased the number of autophagosomes and Beclin1 expression, but attenuated Bcl2 expression. Overexpression of miR-130a decreased the number of autophagosomes, cell death and Beclin1 expression, but promoted Bcl2 expression; these effects were mediated by Runx3. In conclusion, miR-130a is important for maintaining normal autophagy levels and promoting the survival of EPC via regulation of Bcl-2 and Beclin1 expression, via Runx3. MiR-130a may be a regulator linking apoptosis and the autophagy of EPC.

High glucose condition induces autophagy in endothelial progenitor cells contributing to angiogenic impairment.

Cardiovascular complications are the major causes of death in patients with diabetes mellitus. Several studies have demonstrated that endothelial progenitor cells (EPCs), adult stem cells contributing to the regeneration of vascular endothelium, are dysfunctional under diabetic condition resulting in impaired peripheral circulation and delayed wound healing. In this study, we investigated the cellular alteration of EPCs under high glucose condition, to elucidate the mechanisms underlying diabetes-associated EPC dysfunction. EPCs were isolated from bone marrow and cultured in normal glucose (5.5 mM)- or high glucose (HG; 30 mM)-containing medium. High glucose treated-EPCs showed decreased ability to form tubule-like networks in Matrigel compared to EPCs under normal glucose, which matched well to the clinical observation of diabetic EPC dysfunction. Conversion of LC3-I to LC3-II was increased in EPCs under HG condition, showing that HG induced autophagy in EPCs. Flow cytometric analysis revealed generation of oxidative stress and disruption of mitochondrial permeability in HG exposed EPCs. Increased mitochondrial oxidative stress was also observed by mitochondria-specific superoxide indicator, MitoSOX(TM). Taken together, we demonstrated that autophagy and mitochondrial impairment were induced in EPCs under high glucose condition, giving a new insight into the mechanism underlying dysfunction of diabetic EPCs. We hope that our finding can contribute to the development of a new treatment option for cardiovascular complications in diabetic patients.

Autophagy - the friendly fire in endothelial cell regeneration. Focus on “Autophagy in endothelial progenitor cells is cytoprotective in hypoxic conditions”

cardiovascular disease remains the number one killer in the Western world and is growing rapidly in developing countries in Asia and South America (4). While significant progress has been made in understanding the pathogenic process responsible for acute ischemic events such as myocardial infarction and stroke, the cellular response mechanism to hypoxia has remained largely elusive despite extensive investigations in the past three decades. In this issue, Wang et al. (11) identified an essential role of autophagy to protect the survival of endothelial progenitor cells (EPCs) undergoing hypoxia, raising the prospect that modulation of autophagic response in EPCs may provide significant therapeutic advantage following EPCs transplantation.

Autophagy in endothelial progenitor cells is cytoprotective in hypoxic conditions

Endothelial progenitor cells (EPCs) may be incorporated into local vessels to enhance angiogenesis within ischemic tissue. Recently, EPC transplantation has become a potential therapy for improving tissue function in cardiovascular disease. However, the mechanisms of proliferation, differentiation, and survival of EPCs in a hypoxic microenvironment remain unclear. In this study, CD34(+)VEGFR-2(+) EPCs were isolated from mononuclear cells of human umbilical cord blood, and differentiation to endothelial cells was induced with VEGF. When EPC autophagy was inhibited with 3-methyladenine (3-MA) under normoxic conditions, proliferation and viability of the cells were decreased, and the cells failed to differentiate into endothelial cells. Under hypoxic conditions (1% O(2)), Beclin-1 expression of the cells was upregulated and both MDC-labeled and LC3-positive puncta and autophagic ultrastructures in the cells increased significantly. The number of lysosomes also increased in hypoxia-exposed cells. When autophagy was inhibited with 3-MA under hypoxic conditions, the number of apoptotic cells increased, and the number and size of lysosomes decreased. Conversely, apoptosis of the hypoxic EPCs was reduced when autophagy was induced by pretreatment with rapamycin. These results demonstrate that autophagy is involved in proliferation and differentiation of EPCs. Furthermore, hypoxia activates autophagy, promoting EPC survival by inhibiting apoptosis. Enhancing autophagy with hypoxic preconditioning may be beneficial for survival of the transplanted EPCs in a local hypoxic environment.