Proangiogenic microRNAs Research Articles

Extracellular vesicles from differentiated stem cells contain novel proangiogenic miRNAs and induce angiogenic responses at low doses

Extracellular vesicles (EVs) released from healthy endothelial cells (ECs) have shown potential for promoting angiogenesis, but their therapeutic efficacy remains poorly understood. We have previously shown that transplantation of a human embryonic stem cell-derived endothelial cell product (hESC-ECP), promotes new vessel formation in acute ischemic disease in mice, likely via paracrine mechanism(s). Here, we demonstrated that EVs from hESC-ECPs (hESC-eEVs) significantly increased EC tube formation and wound closure invitro at ultralow doses, whereas higher doses were ineffective. More important, EVs isolated from the mesodermal stage of the differentiation (hESC-mEVs) had no effect. Small RNA sequencing revealed that hESC-eEVs have a unique transcriptomic profile and are enriched in known proangiogenic microRNAs(miRNAs, miRs). Moreover, an in silico analysis identified three novel hESC-eEV-miRNAs with potential proangiogenic function. Differential expression analysis suggested that two of those, miR-4496 and miR-4691-5p, are highly enriched in hESC-eEVs. Overexpression of miR-4496 or miR-4691-5p resulted in increased EC tube formation and wound closure invitro, validating the novel proangiogenic function of these miRNAs. In summary, we demonstrated that hESC-eEVs are potent inducers of EC angiogenic response at ultralow doses and contain a unique EV-associated miRNA repertoire, including miR-4496 and miR-4691-5p, with novel proangiogenic function.

Abstract 12931: Development of Novel Targeted, Membrane-Coated Polymer Hybrid Nanoparticles for Endothelial Repair Following Balloon Angioplasty in Rat Carotid Artery Model

Background: Coronary and peripheral arterial disease remains one of the leading causes of death worldwide. Treatments include balloon angioplasty, stent placement, or bypass grafting, all of which damage the endothelium and increase the risk of thrombosis or intimal hyperplasia. Thus, there is a critical need for rapid reendothelialization following these treatments. Endothelial progenitor cell-derived (EPC) extracellular vesicles (EVs) containing proangiogenic microRNAs, such as miR126, is one of the natural mechanisms of vascular repair. However, the translational application of EVs is limited by low EV yield, functional heterogeneity, and difficult tissue targeting. Hypothesis: We hypothesize that EV-inspired hybrid nanoparticles can improve reendothelialization and modification with a specific collagen binding peptide SILY can target vascular injury. Aims: (1) Engineer and characterize a SILY modified hybrid EPC membrane coated nanoparticles (SILY-ENPs) containing a miRNA-126-loaded poly-lactic-co-glycolic acid core (miR126-PLGA) in vitro . (2) Test the therapeutic efficacy of the SILY-ENPs in an in vivo rat carotid balloon angioplasty model. Methods: EPC plasma membranes were isolated from umbilical cord blood EPCs and conjugated with SILY using copper-free Click chemistry. Membrane fragments were coextruded with miR126-PLGA to form SILY-ENPs. Particles were characterized in vitro for collagen and platelet binding and angiogenic function. In vivo function was tested in a rat carotid balloon angioplasty model. Results: SILY-ENPs were found to have increased collagen binding and decreased platelet activity. Delivery of miR126 cargo slightly improved pro-angiogenic protein expression and increased rates of reendothelialization. However, SILY-ENPs were found to stimulate hyperplasia as an adverse side-effect. Conclusions: SILY-ENPs exhibited target specificity, decreased platelet activity, and increased rates of reendothelialization, but increased intimal hyperplasia. This technology represents a novel EV inspired nanoplatform which can be tailored for treating various diseases by altering the cargo or membrane coating.

MicroRNA-216a is essential for cardiac angiogenesis

While it is experimentally supported that impaired myocardial vascularization contributes to a mismatch between myocardial oxygen demand and supply, a mechanistic basis for disruption of coordinated tissue growth and angiogenesis in heart failure remains poorly understood. Silencing strategies that impair microRNA biogenesis have firmly implicated microRNAs in the regulation of angiogenesis, and individual microRNAs prove to be crucial in developmental or tumor angiogenesis. A high-throughput functional screening for the analysis of a whole-genome microRNA silencing library with regard to their phenotypic effect on endothelial cell proliferation as a key parameter, revealed several anti- and pro-proliferative microRNAs. Among those was miR-216a, a pro-angiogenic microRNA which is enriched in cardiac microvascular endothelial cells and reduced in expression under cardiac stress conditions. miR-216a null mice display dramatic cardiac phenotypes related to impaired myocardial vascularization and unbalanced autophagy and inflammation, supporting a model where microRNA regulation of microvascularization impacts the cardiac response to stress.

Abstract 15431: Engineered Nanovesicles With Microrna-126 Cargo Improve Cardiac Repair After Myocardial Infarction in a Rat Ischemia-Reperfusion Model

Introduction: Ckit+ progenitor cells (CPCs) produce small extracellular vesicles (sEVs) which are pivotal for CPC-based cardiac repair after myocardial infarction (MI). sEVs encapsulate potent RNA cargo within a bilayer membrane which aids with sEV uptake. However, despite established benefits, sEV therapies are limited by inconsistent sEV release from cells and variable cargo encapsulation. Further, although synthetic sEV mimics allow cargo tunability, they suffer rapid clearance when administered. We have developed an sEV-like vesicle (ELV) which maintains an sEV-like membrane, important for efficient uptake, but allows cargo control like that of sEV-mimics. We hypothesize that ELVs with proangiogenic microRNA (miR) cargo improve cardiac repair in a rat ischemia-reperfusion (IR) model compared to sEVs and that this occurs in a dose-dependent manner. Methods: sEVs were isolated from CPC conditioned media via ultracentrifugation and sEV inherent cargo was depleted with sonication. ELVs with miR-126 cargo were synthesized from sEVs using square-wave electroporation and characterized. miR-126+ELVs and sEVs (at 5 μg/kg or 10 μg/kg) were administered intramyocardially to the failing left-ventricle (LV) of Sprague Dawley rats after 30-minute IR injury. Infarct size was quantified with TTC/Evans blue staining after 24 hours. LV global and segmental hemodynamic parameters were assessed at baseline, Day 7, 14, and 28. After 28 days, LV fibrosis, hypertrophy and vessel formation were assessed with picrosirius red, wheat germ agglutinin and isolectin staining, respectively. Results: miR-126+ELVs were of similar size and concentration to CPC-sEVs. Upon administration, all groups reduced infarct size 24 hours post-treatment compared to IR-only. Further, ELV groups improved global hemodynamic parameters compared to controls with a dose-dependent segmental response across the LV. Finally, miR-126+ELVs increased vessel density compared to sEVs. Conclusion: ELVs with an sEV-like membrane but tailored cargo allow for cardiac repair after MI in a rat IR model, with both global and regional hemodynamic improvements and tunable histological responses. This work highlights the scope of miR+ELV therapies for cardiac diseases even beyond MI.

RF34 | PMON229 TNFa-Induced Altered miRNA Expression Links to Inflammation in Endometriosis

Abstract Endometriosis is a common gynecological inflammatory disorder, which is characterized by immune system dysregulation with initiation and progression. It affects 5% to 15% of reproductive-age women and is present in as many as 30% to 50% of patients with infertility and/or pain. In previous studies, including ours, have demonstrated that several cytokines have been associated with the evolution of endometriosis, including tumor necrosis factor-a (TNFa). TNFa is a non-glycosylated protein which has potent inflammatory, cytotoxic, and angiogenic potential. Therefore, in the current studies, we examined the effects of TNFa over a time course in the regulation of proinflammatory and proangiogenic microRNAs (miRNAs) in primary cultures of normal endometrial stromal cells (NESC) and compared with the untreated cells derived from eutopic endometrium of endometriosis subjects (EESC). miRNAs are short, 18- to 22-nucleotide– size, non-coding RNAs that act as post-transcriptional modulators of gene expression and are involved in the pathogenesis of endometriosis. Using NanoString nCounter-based assays and quantitative RT-PCR, we have identified levels of several proinflammatory and proangiogenic miRNAs higher in EESC than NESC. NESC treatment with TNFa significantly altered the expression of proinflammatory and proangiogenic miRNAs in a time-dependent manner. Notably, TNFa significantly decreased phosphorylation of the PI3K, AKT, and ERK signaling pathways. Moreover, treatment of EESC and NESC with curcumin (diferuloylmethane, CUR), an anti-inflammatory folk medicine in Asian countries, significantly increased the expression of anti-inflammatory and anti-angiogenic miRNAs in a dose- and time-dependent manner. These findings demonstrate higher inflammatory, and proangiogenic miRNA production in EESC may be due to a higher concentration of TNFa than NESC under basal conditions. Therefore, suppressing TNFa may reduce the inflammatory and angiogenic miRNA associated with endometriosis. Sources of Research Support: This study was supported in part by National Institutes of Health Grants 1SC3 GM113751, U01, 1SC1 GM130544, HD66439, 1R01HD057235, U54 CA118948, HD41749, S21MD000101 and G12-MD007602. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Grant #C06 RR018386 from NIH/NCRR. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 1:06 p.m. - 1:11 p.m.

Potential of Circulating Proangiogenic MicroRNAs for Predicting Major Adverse Cardiac and Cerebrovascular Events in Unprotected Left Main Coronary Artery Disease Patients Who Underwent Coronary Artery Bypass Grafting

Objective: This study aimed to explore the association of 14 proangiogenic microRNAs (miRNAs) with major adverse cardiac and cerebrovascular events (MACCE) occurrence in unprotected left main coronary artery disease (ULMCAD) patients who underwent coronary artery bypass grafting (CABG). Methods: A total of 196 ULMCAD patients who underwent first ever CABG were recruited. The peripheral blood samples were collected prior to CABG, and then plasma samples were separated to detect expressions of 14 proangiogenic miRNAs by the reverse transcription quantitative PCR. Patients were regularly followed up to MACCE occurrence or 36 months after CABG. Results: MACCE occurrence at 1 year, 2 years, and 3 years was 7.1, 11.2, and 14.3%, respectively, and accumulating MACCE occurrence time was 32.7 (95% confidence interval: 31.5–33.9) months. Both Kaplan-Meier curves and univariate Cox’s regression analyses displayed that miR-let-7f, miR-19a, miR-126, miR-130a, and miR-378 high expressions were associated with lower accumulating MACCE occurrence. Furthermore, forward stepwise multivariate Cox’s regression disclosed that miR-let-7f high expression and miR-378 high expression independently predicted decreased accumulating MACCE occurrence, whereas BMI (>25.0 kg/m²), diabetes, previous stroke, and higher disease extent were independent predictive factors for elevated accumulating MACCE occurrence. Conclusion: Measurement of circulating proangiogenic miRNAs especially miR-let-7f, miR-19a, miR-126, miR-130a, and miR-378 helps predict MACCE risk in ULMCAD patients who underwent CABG.

Exosomes secreted from mesenchymal stem cells mediate the regeneration of endothelial cells treated with rapamycin by delivering pro-angiogenic microRNAs

Delayed endothelial healing after drug eluting stent (DES) implantation is a critical clinical problem in treatment of coronary artery diseases. Exosomes exhibit proangiogenic potential in a variety of ischemic diseases. However, the association of exosomes with endothelial regeneration after DES implantation has been rarely reported. In this study, we aimed to investigate the therapeutic effects of mesenchymal stem cell (MSC)-derived exosomes on endothelial cells treated with rapamycin and explore the potential mechanisms of MSC-derived exosomes in promoting endothelial regeneration. Exosomes were isolated from MSCs by ultracentrifugation and identified by transmission electron microscopy, nanoparticle tracking analysis, and Western blot assay. The in vitro effects of MSC-derived exosomes on the proliferation and migration of endothelial cells treated with rapamycin were evaluated by integrated experiment, cell counting kit-8, scratch, tube formation, and transwell assays. And the apoptosis of rapamycin-induced endothelial cells loaded with MSC-derived exosomes was detected using TUNEL and Annexin-V FITC and PI double-staining assays. The microRNA (miRNA) cargo of MSC-derived exosomes was identified by high-throughput RNA sequencing. Pro-angiogenic miRNAs and key pathways were further characterized. Our results indicated that MSC-derived exosomes could be ingested into umbilical vein endothelial cells (HUVECs) and significantly enhanced cell proliferation rate, migratory and tube-forming capabilities in vitro. MSC-derived exosomes also inhibited the apoptosis of HUVECs induced by rapamycin. A distinct class of exosomal miRNAs was further identified, including six miRNAs tightly related to neovasculogenesis. Silencing the expression of exosomal miRNA-21–5p and let-7c-5p attenuated the pro-proliferative and pro-migratory capacity of MSC-derived exosomes. Moreover, functional enrichment analysis indicated that metabolic pathways might contribute to reendothelialization. This study highlights a proregenerative effect of MSC-derived exosomes in vitro, which may be partly explained by the delivery of pro-angiogenic miRNAs to endothelial cells.

Proangiogenic signature in advanced laryngeal carcinoma after microRNA expression profiling.

The aim of this study is to evaluate which dysregulated angiomiRs compose the specific proangiogenic microRNA signature of advanced laryngeal cancer and review the literature. Thirty-six samples from twelve patients with advanced laryngeal carcinoma were collected. Total RNA was extracted and microRNA global profiling was performed using Agilent Technologies Microarray Kit. Fifty-nine microRNAs were found to have significantly different expression levels. Eleven microRNAs from the whole group were sorted as regulators of tumor angiogenesis (angiomiRs): seven were up-regulated-miR-1246, miR-181b 5p, miR-18a 5p, miR-21 3p, miR-210 3p, miR-503 5p, miR-93 5p and four were down-regulated-miR148a 5p, miR-145 5p, miR-204 5p, miR-125b 5p. For none of those microRNAs we found heterogeneity in tumor tissue. We are the first to report the specific proangiogenic microRNA signature in advanced laryngeal carcinoma and we confirm and amplify findings from previous studies that expand our perception of a specific "molecular state" of angiogenesis that is distinctive only for laryngeal cancer.

Reduced expression of microRNA-130a promotes endothelial cell senescence and age-dependent impairment of neovascularization.

Aging is associated with impaired neovascularization in response to ischemia. MicroRNAs are small noncoding RNAs emerging as key regulators of physiological and pathological processes. Here we investigated the potential role of microRNAs in endothelial cell senescence and age-dependent impairment of neovascularization. Next generation sequencing and qRT-PCR analyses identified miR-130a as a pro-angiogenic microRNA which expression is significantly reduced in old mouse aortic endothelial cells (ECs). Transfection of young ECs with a miR-130a inhibitor leads to accelerated senescence and reduced angiogenic functions. Conversely, forced expression of miR-130a in old ECs reduces senescence and improves angiogenesis. In a mouse model of hindlimb ischemia, intramuscular injection of miR-130a mimic in older mice restores blood flow recovery and vascular densities in ischemic muscles, improves mobility and reduces tissue damage. miR-130a directly targets antiangiogenic homeobox genes MEOX2 and HOXA5. MEOX2 and HOXA5 are significantly increased in the ischemic muscles of aging mice, but forced expression of miR-130a reduces the expression of these factors. miR-130a treatment after ischemia is also associated with increased number and improved functional activities of pro-angiogenic cells (PACs). Forced expression of miR-130a could constitute a novel strategy to improve blood flow recovery and reduce ischemia in older patients with ischemic vascular diseases.

MiRNA profiling in renal carcinoma suggest the existence of a group of pro-angionenic tumors in localized clear cell renal carcinoma.

Renal cell carcinoma comprises a variety of entities, the most common being the clear-cell, papillary and chromophobe subtypes. These subtypes are related to different clinical evolution; however, most therapies have been developed for clear-cell carcinoma and there is not a specific treatment based on different subtypes. In this study, one hundred and sixty-four paraffin samples from primary nephrectomies for localized tumors were analyzed. MiRNAs were isolated and measured by microRNA arrays. Significance Analysis of Microarrays and Consensus Cluster algorithm were used to characterize different renal subtypes. The analyses showed that chromophobe renal tumors are a homogeneous group characterized by an overexpression of miR 1229, miR 10a, miR 182, miR 1208, miR 222, miR 221, miR 891b, miR 629-5p and miR 221-5p. On the other hand, clear cell renal carcinomas presented two different groups inside this histological subtype, with differences in miRNAs that regulate focal adhesion, transcription, apoptosis and angiogenesis processes. Specifically, one of the defined groups had an overexpression of proangiogenic microRNAs miR185, miR126 and miR130a. In conclusion, differences in miRNA expression profiles between histological renal subtypes were established. In addition, clear cell renal carcinomas had different expression of proangiogenic miRNAs. With the emergence of antiangiogenic drugs, these differences could be used as therapeutic targets in the future or as a selection method for tailoring personalized treatments.

Potential of circulating pro-angiogenic microRNA expressions as biomarkers for rapid angiographic stenotic progression and restenosis risks in coronary artery disease patients underwent percutaneous coronary intervention.

BackgroundThis study aimed to investigate the correlation of pro‐angiogenic microRNA (miRNA) expressions with rapid angiographic stenotic progression (RASP) and restenosis risks in coronary artery disease (CAD) patients underwent percutaneous coronary intervention (PCI) with drug‐eluting stents (DES).MethodsA total of 286 CAD patients underwent PCI with DES were consecutively recruited in this study. Plasma samples were collected before PCI operation, and 14 pro‐angiogenic miRNAs were measured by real‐time quantitative reverse transcription‐polymerase chain reaction. Rapid angiographic stenotic progression at nontarget lesions and restenosis at stented lesions were evaluated by quantitative coronary angiography at 12 months after PCI operation.ResultsThe occurrence rates of RASP and restenosis were 39.5% and 22.4%, respectively. Let‐7f, miR‐19a, miR‐19b‐1, miR‐92a, miR‐126, miR‐210, and miR‐296 were decreased in RASP patients than non‐RASP patients, among which let‐7f, miR‐19a, miR‐126, miR‐210, and miR‐296 independently correlated with lower RASP occurrence by multivariate analysis, followed by receiver‐operating characteristic (ROC) curve exhibited that these five miRNAs showed great value in predicting RASP risk with area under curve (AUC) 0.879 (95% CI: 0.841‐0.917). Besides, let‐7f, miR‐19a, miR‐92a, miR‐126, miR‐130a, and miR‐210 were reduced in restenosis patients than non‐restenosis patients, among them miR‐19a, miR‐126, miR‐210, and miR‐378 independently correlated with lower restenosis occurrence by multivariate analysis, followed by ROC curve disclosed that these four miRNAs had good value in predicting restenosis risk with AUC 0.776 (95% CI: 0.722‐0.831).ConclusionsCirculating let‐7f, miR‐19a, miR‐126, miR‐210, and miR‐296 independently correlate with reduced RASP risk, while miR‐19a, miR‐126, miR‐210, and miR‐378 independently correlate with decreased restenosis risk in CAD patients underwent PCI with DES.

SAT-200 Therapeutic Targeting of Functional MicroRNA Expression by Curcumin in Human Endometrial Stromal Cells

Endometriosis is a chronic gynecological inflammatory disorder in which immune system dysregulation is thought to play a role in its initiation and progression. Current agency-approved hormonal therapies, including synthetic progestins, GnRH-agonists, and danazol are often of limited efficacy and counterproductive to fertility, and cause systemic side effects due to suppression of endogenous steroid hormone production. Our recent publication (PMID: 30259980) suggests that curcumin (diferuloylmethane, CUR), an anti-inflammatory folk medicine in Asian countries, has therapeutic potential to reduce inflammation associated with endometriosis. Therefore, in the current studies we examined the effects of CUR at different doses over a time course in the regulation of proinflammatory and proangiogenic microRNAs (miRNAs) in primary cultures of normal endometrial stromal cells (NESC) and cells derived from eutopic endometrium of endometriosis subjects (EESC). miRNAs are non-coding RNAs that regulate protein translation and have been shown to be involved in the pathogenesis of endometriosis. Using NanoString nCounter-based assays and semi-quantitative RT-PCR we have identified levels of several proinflammatory and proangiogenic miRNAs (including has-miR-196b-5p, has-miR-199a-5p, has-miR-21-5p) that are higher in EESC compared to NESC. EESC and NESC treatment with CUR significantly reduced expression of proinflammatory and proangiogenic miRNAs in a dose- and time-dependent manner. Notably, CUR significantly decreased phosphorylation of the AKT, ERK and prohibitin signaling pathways. These findings demonstrate higher proinflammatory and proangiogenic miRNA production in EESC compared to NESC under basal conditions, and suggest that by suppressing these factors, CUR has the therapeutic potential to reduce inflammation associated with endometriosis. Nothing to Disclose: IC; SB; AD; WX; CN; NS, RNT, WET. Sources of Research Support: This study was supported in part by National Institutes of Health Grants 1SC3 GM113751, U01 HD66439, 1R01HD057235, U54 CA118948, HD41749, S21MD000101 and G12-MD007602. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Grant #C06 RR018386 from NIH/NCRR.

Extracellular Vesicles From Human Urine-Derived Stem Cells Ameliorate Erectile Dysfunction in a Diabetic Rat Model by Delivering Proangiogenic MicroRNA.

IntroductionStem cell therapies represent a promising new frontier for the treatment of refractory diabetic erectile dysfunction (DED). The use of stem cell-derived extracellular vesicles (EVs) is a novel strategy for cell-free stem cell therapy. We have reported that urine-derived stem cells (USCs) can improve DED; however, the therapeutic effects of EVs secreted by USCs (USC-EVs) remain unknown.AimTo determine the therapeutic effects of USC-EVs on DED in a rat model.MethodsUSC-EVs were isolated from conditioned medium by ultracentrifugation. DED was induced in male Sprague–Dawley rats via an intraperitoneal injection of streptozotocin. Sixteen DED rats were divided into phosphate-buffered saline (PBS) and USC-EV groups. Eight normal rats served as the normal control group. PBS or USC-EVs were transplanted into the corpora cavernosa in the corresponding groups.Main Outcome MeasureIntracavernosal pressure (ICP), mean arterial pressure (MAP), expression of endothelial markers (CD31), endothelial nitric oxide synthase (eNOS), phospho-eNOS, and neural nitric oxide synthase (nNOS) were assessed in each group. Masson’s trichrome staining was used to determine the collagen deposition and ratio of smooth muscle cells to collagen. The microRNA (miRNA) cargo of USC-EVs was characterized by high-throughput RNA sequencing.ResultsRecovery of erectile function was observed in the USC-EV group, as represented by improved ICP and ICP/MAP ratio. CD31, eNOS, phospho-eNOS, and nNOS expression in the penis was significantly improved in the USC-EV group. In addition, the ratio of smooth muscle to collagen was significantly increased in the USC-EV group. RNA sequencing revealed that USC-EVs were enriched for distinct classes of miRNA (miR-21-5p, let-7 family, miR-10 family, miR-30 family, and miR-148a-3p) that promote angiogenesis.ConclusionUSC-EV transplantation can ameliorate DED in rats. Its mechanism may involve the delivery of proangiogenic miRNA.Ouyang B, Xie Y, Zhang C, et al. Extracellular Vesicles From Human Urine-Derived Stem Cells Ameliorate Erectile Dysfunction in a Diabetic Rat Model by Delivering Proangiogenic MicroRNA. Sex Med 2019;7:241–250.

Therapeutic regulation of VE-cadherin with a novel oligonucleotide drug for diabetic eye complications using retinopathy mouse models.

A major feature of diabetic retinopathy is breakdown of the blood-retinal barrier, resulting in macular oedema. We have developed a novel oligonucleotide-based drug, CD5-2, that specifically increases expression of the key junctional protein involved in barrier integrity in endothelial cells, vascular-endothelial-specific cadherin (VE-cadherin). CD5-2 prevents the mRNA silencing by the pro-angiogenic microRNA, miR-27a. CD5-2 was evaluated in animal models of ocular neovascularisation and vascular leak to determine its potential efficacy for diabetic retinopathy. CD5-2 was tested in three mouse models of retinal dysfunction: conditional Müller cell depletion, streptozotocin-induced diabetes and oxygen-induced retinopathy. Vascular permeability in the Müller cell-knockout model was assessed by fluorescein angiography. The Evans Blue leakage method was used to determine vascular permeability in streptozotocin- and oxygen-induced retinopathy models. The effects of CD5-2 on retinal neovascularisation, inter-endothelial junctions and pericyte coverage in streptozotocin- and oxygen-induced retinopathy models were determined by staining for isolectin-B4, VE-cadherin and neural/glial antigen 2 (NG2). Blockmir CD5-2 localisation in diseased retina was determined using fluorescent in situ hybridisation. The effects of CD5-2 on VE-cadherin expression and in diabetic retinopathy-associated pathways, such as the transforming growth factor beta (TGF-β) and wingless/integrated (WNT) pathway, were confirmed using western blot of lysates from HUVECs, a mouse brain endothelial cell line and a VE-cadherin null mouse endothelial cell line. CD5-2 penetrated the vasculature of the eye in the oxygen-induced retinopathy model. Treatment of diseased mice with CD5-2 resulted in reduced vascular leak in all three animal models, enhanced expression of VE-cadherin in the microvessels of the eye and improved pericyte coverage of the retinal vasculature in streptozotocin-induced diabetic models and oxygen-induced retinopathy models. Further, CD5-2 reduced the activation of retinal microglial cells in the streptozotocin-induced diabetic model. The positive effects of CD5-2 seen in vivo were further confirmed in vitro by increased protein expression of VE-cadherin, SMAD2/3 activity, and platelet-derived growth factor B (PDGF-B). CD5-2 has therapeutic potential for individuals with vascular-leak-associated retinal diseases based on its ease of delivery and its ability to reverse vascular dysfunction and inflammatory aspects in three animal models of retinopathy.

Adenosine-to-Inosine Editing of MicroRNA-487b Alters Target Gene Selection After Ischemia and Promotes Neovascularization.

Adenosine-to-inosine editing of microRNAs has the potential to cause a shift in target site selection. 2'-O-ribose-methylation of adenosine residues, however, has been shown to inhibit adenosine-to-inosine editing. To investigate whether angiomiR miR487b is subject to adenosine-to-inosine editing or 2'-O-ribose-methylation during neovascularization. Complementary DNA was prepared from C57BL/6-mice subjected to hindlimb ischemia. Using Sanger sequencing and endonuclease digestion, we identified and validated adenosine-to-inosine editing of the miR487b seed sequence. In the gastrocnemius muscle, pri-miR487b editing increased from 6.7±0.4% before to 11.7±1.6% (P=0.02) 1 day after ischemia. Edited pri-miR487b is processed into a novel microRNA, edited miR487b, which is also upregulated after ischemia. We confirmed editing of miR487b in multiple human primary vascular cell types. Short interfering RNA-mediated knockdown demonstrated that editing is adenosine deaminase acting on RNA 1 and 2 dependent. Using reverse-transcription at low dNTP concentrations followed by quantitative-PCR, we found that the same adenosine residue is methylated in mice and human primary cells. In the murine gastrocnemius, the estimated methylation fraction increased from 32.8±14% before to 53.6±12% 1 day after ischemia. Short interfering RNA knockdown confirmed that methylation is fibrillarin dependent. Although we could not confirm that methylation directly inhibits editing, we do show that adenosine deaminase acting on RNA 1 and 2 and fibrillarin negatively influence each other's expression. Using multiple luciferase reporter gene assays, we could demonstrate that editing results in a complete switch of target site selection. In human primary cells, we confirmed the shift in miR487b targeting after editing, resulting in a edited miR487b targetome that is enriched for multiple proangiogenic pathways. Furthermore, overexpression of edited miR487b, but not wild-type miR487b, stimulates angiogenesis in both in vitro and ex vivo assays. MiR487b is edited in the seed sequence in mice and humans, resulting in a novel, proangiogenic microRNA with a unique targetome. The rate of miR487b editing, as well as 2'-O-ribose-methylation, is increased in murine muscle tissue during postischemic neovascularization. Our findings suggest miR487b editing plays an intricate role in postischemic neovascularization.

Beneficial effects of exosomes secreted by cardiac-derived progenitor cells and other cell types in myocardial ischemia.

When injected into acutely infarcted rodent or pig hearts, naturally secreted nanovesicles known as exosomes from cardiac-derived progenitor cells (CPCs) reduce scar size and improve cardiac function. In this regard, exosomes fully mimic the benefits of injecting their parent cells. This recognition paves the way to the development of exosome-based, cell-free treatments for heart disease that could possibly supplant cell-based therapies. Mechanisms of benefit of these vesicles are incompletely understood but cytoprotection, stimulation of angiogenesis, induction of antifibrotic cardiac fibroblasts, and modulation of M1/M2 polarization of macrophages infiltrating the infarcted region can all play important roles. Accordingly, the beneficial molecules carried by CPC-secreted exosomes have been identified only in part but cytoprotective and proangiogenic microRNAs (miRNA) and proteins have been described. Besides CPC-secreted exosomes, vesicles released from other cell types including mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iSPCs) have also been associated with cardioprotection. This review aims to discuss recent advances in our understanding of the role of secreted vesicles in cardiac repair, with a focus on CPC-derived exosomes.

Transplantation of adipose tissue-derived stem cell-derived exosomes ameliorates erectile function in diabetic rats.

Mesenchymal stem cells (MSCs) have been considered as an attractive tool for the therapy of diseases. Accumulating evidence indicates that the healing effects of MSCs are mainly related to paracrine action rather than transdifferentiation. Exosomes excreted from MSCs have emerged as physiologically relevant and powerful components of the MSC secretome. However, whether MSC-derived exosomes can improve erectile function of streptozotocin-induced diabetic rats and its mechanism remains unknown. Our previous work showed that adipose tissue-derived stem cells (ADSCs) transplantation could increase endothelial and smooth muscle contents and improve erectile function of diabetic rats. In this study, ADSC-derived exosomes (ADSC-Exo) exhibited in vitro proangiogenic properties, induced the proliferation of endothelial cells and restored erectile function in vivo, as well as decreased fibrosis of corpus cavernosum. In further experiments, we found that ADSC-Exo contained some proangiogenic (miR-126, miR-130a and miR-132) microRNAs and an antifibrotic microRNA family (miR-let7b and miR-let7c). Thus, it is reasonable to postulate that ADSC-Exo transports key functional miRNAs to target cells in a specific manner to improve functional recovery or to activate endogenous repair mechanisms. This proof-of-concept study provides a novel approach for the treatment of diabetic erectile dysfunction.

MicroRNA-150 Modulates Ischemia-Induced Neovascularization in Atherosclerotic Conditions.

Hypercholesterolemia is an atherosclerotic condition that is associated with impaired neovascularization in response to ischemia. This study sought to define the role of microRNAs in that pathophysiology. Next-generation sequencing and quantitative reverse transcription polymerase chain reaction analyses identified miR-150 as a proangiogenic microRNA, which expression is significantly reduced in the ischemic muscles of hypercholesterolemic apolipoprotein E-deficient (ApoE-/-) mice, and in human umbilical vein endothelial cells exposed to oxidized low-density lipoprotein. Forced expression of miR-150 using a miR mimic could rescue oxidized low-density lipoprotein-mediated impairment of endothelial cell migration and tubule formation in vitro. In a mouse model of hindlimb ischemia, intramuscular injection of miR-150 mimic restored blood flow recuperation, vascular densities in ischemic muscles, and functional mobility in ApoE-/- mice. Treatment of ApoE-/- mice with miR-150 also increased the number and the activities of proangiogenic cells. miR-150 targets SRC kinase signaling inhibitor 1, an important regulator of Src (proto-oncogene tyrosine-protein kinase Src) activity. Here we found that hypercholesterolemia and oxidized low-density lipoprotein exposure are associated with increased SRC kinase signaling inhibitor 1 expression and decreased Src activity. However, treatment with miR-150 mimic reduces SRC kinase signaling inhibitor 1 expression and restores Src and downstream endothelial nitric oxide synthase and Akt (protein kinase B) activities both in vitro and in vivo. We also demonstrate the interrelation between miR-150 and SRC kinase signaling inhibitor 1 and their importance for endothelial cell angiogenic activities. Hypercholesterolemia is associated with reduced expression of miR-150, impaired Src signaling, and inefficient neovascularization in response to ischemia. Forced expression of miR-150 using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions.

Endothelial progenitor cells and cardiovascular risk: does ageing trump all other factors?

Discovered nearly 20 years ago, endothelial progenitor cells (EPC) attract both basic and clinical researchers (1). As key regulators of vascular homeostasis in health and disease, circulating progenitor cell levels reflect endogenous regenerative potential. Upon endothelial damage, EPC are mobilized from the bone marrow and follow chemokine gradients to sites of endothelial injury where they assist in endothelial repair mainly via paracrine mechanisms. Next to the production of angiogenic growth factors, novel modes of paracrine regulation are being discovered, such as the release of endothelial cell-derived microparticles or microvesicles that contain pro-angiogenic microRNAs (2).

Diverse impact of xeno-free conditions on biological and regenerative properties of hUC-MSCs and their extracellular vesicles

Growing evidence indicates that intracellular signaling mediated by extracellular vesicles (EVs) released by stem cells plays a considerable role in triggering the regenerative program upon transplantation. EVs from umbilical cord mesenchymal stem cells (UC-MSC-EVs) have been shown to enhance tissue repair in animal models. However, translating such results into clinical practice requires optimized EV collection procedures devoid of animal-originating agents. Thus, in this study, we analyzed the influence of xeno-free expansion media on biological properties of UC-MSCs and UC-MSC-EVs for future applications in cardiac repair in humans. Our results show that proliferation, differentiation, phenotype stability, and cytokine secretion by UC-MSCs vary depending on the type of xeno-free media. Importantly, we found distinct molecular and functional properties of xeno-free UC-MSC-EVs including enhanced cardiomyogenic and angiogenic potential impacting on target cells, which may be explained by elevated concentration of several pro-cardiogenic and pro-angiogenic microRNA (miRNAs) present in the EVs. Our data also suggest predominantly low immunogenic capacity of certain xeno-free UC-MSC-EVs reflected by their inhibitory effect on proliferation of immune cells in vitro. Summarizing, conscious selection of cell culture conditions is required to harvest UC-MSC-EVs with the optimal desired properties including enhanced cardiac and angiogenic capacity, suitable for tissue regeneration.Key messageType of xeno-free media influences biological properties of UC-MSCs in vitro.Certain xeno-free media promote proliferation and differentiation ability of UC-MSCs.EVs collected from xeno-free cultures of UC-MSCs are biologically active.Xeno-free UC-MSC-EVs enhance cardiac and angiogenic potential of target cells.Type of xeno-free media determines immunomodulatory effects mediated by UC-MSC-EVs.