Human Endothelial Progenitor Cells Research Articles

Phenotypic characteristics of human bone marrow-derived endothelial progenitor cells in vitro support cell effectiveness for repair of the blood-spinal cord barrier in ALS

Amyotrophic lateral sclerosis (ALS) was recently recognized as a neurovascular disease. Accumulating evidence demonstrated blood-spinal-cord barrier (BSCB) impairment mainly via endothelial cell (EC) degeneration in ALS patients and animal models. BSCB repair may be a therapeutic approach for ALS. We showed benefits of human bone marrow endothelial progenitor cell (hBMEPC) transplantation into symptomatic ALS mice on barrier restoration; however, cellular mechanisms remain unclear. The study aimed to characterize hBMEPCs in vitro under normogenic conditions. hBMEPCs were cultured at different time points. Enzyme-linked immunosorbent assay (ELISA) was used to detect concentrations of angiogenic factors (VEGF-A, angiogenin-1, and endoglin) and angiogenic inhibitor endostatin in conditioned media. Double immunocytochemical staining for CD105, ZO-1, and occludin with F-actin was performed. Results showed predominantly gradual significant post-culture increases of VEGF-A and angiogenin-1 levels. Cultured cells displayed distinct rounded or elongated cellular morphologies and positively immunoexpressed for CD105, indicating EC phenotype. Cytoskeletal F-actin filaments were re-arranged according to cell morphologies. Immunopositive expressions for ZO-1 were detected near inner cell membrane and for occludin on cell membrane surface of adjacent hBMEPCs. Together, secretion of angiogenic factors by cultured cells provides evidence for a potential mechanism underlying endogenous EC repair in ALS through hBMEPC transplantation, leading to restored barrier integrity. Also, ZO-1 and occludin immunoexpressions, confirming hBMEPC interactions in vitro, may reflect post-transplant cell actions in vivo.

Mitochondrial fission protein 1 up-regulation ameliorates senescence-related endothelial dysfunction of human endothelial progenitor cells.

We investigated the contribution of mitochondrial dysfunction to the senescence of human endothelial progenitor cells (EPCs) expanded in vitro and the underlying molecular mechanism. Serial passage increased cell doubling time and those cells reaching the doubling time for more than 100% were defined as senescent EPCs, of which the activity of therapeutic angiogenesis was attenuated in mouse ischemic hindlimbs. The senescent cells, in medium free of glucose and bicarbonate, showed impaired activity in migration and tube formation. Flow cytometry indicated increased content of reactive oxygen species, mitochondria, and calcium, while bioenergetic analysis showed increased oxygen consumption and reduced ATP content. Examination of mitochondrial network showed that senescence increased the length of the network and ultrastructure analysis exhibited elongated mitochondria. Immunoblotting of the senescent EPCs demonstrated decreased expression level of fission protein1 (Fis1). In rat EPCs, the Fis1 level was decreased in the animals aged 24months or older, compared to those of 3months. Silencing of Fis1 in the young EPCs using Fis1-specific siRNA leads to appearance of phenotype resembling those of senescent cells, including elevated oxidative stress, disturbed mitochondrial network, reduced mitochondria membrane potential, decreasing ATP content, lower proliferation activity, and loss of therapeutic potential in ischemic hindlimbs. Fis1 over-expression in senescent EPCs reduced the oxidative stress, increased the proliferation, and restored the cobble stone-like morphology, senescence, bioenergetics, angiogenic potential, and therapeutic activity. In human EPCs, down-regulation of Fis1 is involved in mitochondrial dysfunction and contributes to the impaired activity of EPCs during the senescence process. Enhanced expression of Fis1 in senescent EPCs restores the youthful phenotype.

YBX-1 mediated sorting of miR-133 into hypoxia/reoxygenation-induced EPC-derived exosomes to increase fibroblast angiogenesis and MEndoT

BackgroundMyocardial fibrosis is a common pathophysiological change in cardiovascular disease, which can cause cardiac dysfunction and even sudden death. Excessively activated fibroblasts proliferate and secret excessive extracellular matrix (ECM) components, resulting in normal cardiac structural damage and cardiac fibrosis. We previously found that human endothelial progenitor cell (EPC)-derived exosomes, after hypoxia/reoxygenation (H/R) induction, could significantly increase the mesenchymal-endothelial transition (MEndoT) compared to normal culture EPC-derived exosomes. Exosomes have been shown to carry different nucleic acids, including microRNAs. However, the effects of microRNAs in EPC-derived exosomes on MEndoT and myocardial fibrosis remain unknown.MethodsEPCs were isolated from human peripheral blood, and fibroblasts were isolated from rat hearts, then transfected with miR-133 inhibitor, si-YBX-1, and ov-YBX-1 into EPCs. After H/R induction for 48 h, isolation and characterization of exosomes derived from human EPCs were performed. Finally, fibroblasts were treated by exosome at 48 h. The expression of miR-133 was measured by qRT-PCR; YBX-1 expression was measured by qRT-PCR and western blot. Angiopoiesis was measured by tube formation assay. Endothelial markers and fibrosis markers were measured by western blot.ResultsH/R treatment promoted miR-133 expression in EPCs and EPC-derived exosomes. miR-133 could be incorporated into exosomes and transmitted to cardiac fibroblasts, increasing the angiogenesis and MEndoT of cardiac fibroblasts. miR-133 silencing in H/R-induced EPCs could inhibit miR-133 expression in EPCs and EPCs-derived exosomes. miR-133 silencing in H/R-induced EPCs could inhibit the angiogenesis and MEndoT of cardiac fibroblasts and reverse the effect of H/R treatment. Additionally, miR-133 was specially sorted into H/R-induced EPC-derived exosomes via YBX-1. YBX-1 silencing inhibited miR-133 transfer and reduced fibroblast angiogenesis and MEndoT.ConclusionmiR-133 was specially sorted into H/R-induced EPC-derived exosomes via YBX-1 to increase fibroblast angiogenesis and MEndoT.

Effects of astragaloside IV on activity and proliferative function of endothelial progenitor cells

Objective The objective is to probe into the effects of astragaloside Ⅳ (AS-Ⅳ) on activity and proliferative function of endothelial progenitor cells (EPCs), which lays a foundation for further study on the effects of AS-Ⅳ on vascular neovascularization mediated by endothelial progenitor cells. Methods The mononuclear cells were isolated by the density gradient centrifugation in umbilical cord blood of full-term healthy infants, and EPCs were obtained by subculture and cell identification when the cells presented spindle shapes. The obtained EPCs were randomly divided into the experimental group and the control group. In the experimental group, EPCs were cultured by AS-Ⅳ with different concentration gradients (25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L), while in the control group, they were treated with the same amount of phosphate buffer saline (PBS) solutions. The effects of AS-Ⅳ on the proliferation of endothelial progenitor cells was studied by cell counting kit-8 (CCK-8) cell proliferation experiment, and the activity rate of EPCs cells was measured at the optimum concentration of EPCs proliferation. Results EPCs were successfully obtained after confirming nuclear staining test of CD31 antibody and 4′, 6-diamidino-2-phenylindole (DAPI). Further study showed that AS-Ⅳ can promote the proliferation of EPCs, and its optimal concentration of EPCs proliferation is 100 mg/L. Compared with the normal control group, the activity rate of endothelial progenitor cells after intervention of AS-Ⅳ was 98.7%, higher than 98.12% in the control group, with significant difference (χ2=49.59, P<0.01). Conclusions AS-Ⅳ can enhance the activity of human EPCs and promote their proliferation in vitro. Key words: Astragalus membranaceus; Saponins; Endothelial progenitor cells; Cell proliferation; Cell survival; In vitro

Abstract 410: A Novel Combination Therapy Using First Trimester Human Umbilical Cord-Derived Mesenchymal Stromal Cells and Endothelial Progenitor Cells Significantly Improves Angiogenesis and Cardiac Recovery Following Myocardial Infarction

Introduction: Umbilical cord-derived MSC show pericyte-like characteristics and demonstrate significant angiogenic potential via paracrine and direct support to the vasculature. Endothelial progenitor cells (EPC) have the potential to initiate vascular structures while maintaining paracrine properties following ischemic injury. Hypothesis: A combination therapy of first trimester umbilical cord MSC (FTM HUCPVC) with rat EPC will promote significant angiogenesis, cardiomyocyte survival and lead to functional cardiac recovery in a rat model of MI. Methods: MI was induced by LAD ligation on 8-week-old Fox rnu rats. 1 week later, rats with notable decrease (&lt;30%) in ejection fraction (EF) were randomly separated into 4 treatment groups (n=9) and received intramyocardial injection of: G1: Media; G2: FTM HUPVC (3х10 6 cells/rat); G3: EPC (2.2х10 6 cells/rat); G4: FTM HUCPVC - EPC combination (1:2, total cells 3х10 6 cells/rat). Endpoint analysis was at 5-days and 4-weeks after injection. Results: 5-days after injection, exclusively G4 animals showed significant cardiac recovery, improved EF and FS compared to G1 - G2 - G3 ( p &lt;0.01). IHC analysis showed significantly less apoptosis (caspase-3), more protease activity and sarcomeric actin in combination groups G4 compared to G1-G2-G3 ( p &lt;0.001). More FTM HUCPVC and EPC colocalized adjunct to small capillaries in G4 . Significant increase of human Angpt-2 was detected in G4 compared to G1-G2-G3 (p&lt;0.0001). Analysis at 4-weeks post-injection showed significant improvements in end-systolic volume (128μl) and EF (43%) in G4 compared to G2 (160μl) (35%) G3 (180μl) (30%) respectively (B) . LV contractility (dpdt, tau) significantly improved in G4 compared to G1 , G2 , G3 (p&lt;0.001). Significant reduction in scar tissue and increased LV myocardial mass was found in G4 and G2. IHC analysis showed significantly more and larger (9um) capillaries localized in LAD injured myocardium in G4 compared to G1-G2-G3 ( p &lt;0.001). Significantly more sarcomeric actinin and connexin-43 was identified in G4 compared to G1-G2-G3 . Conclusions: Our results show the superior potential of FTM HUCPVC - EPC combination cell therapy for faster and greater cardiovascular repair and recovery compared to single-cell-type treatments for MI.

Abstract 708: Platelet Derived Growth Factor-Beta Communication Axis is Critical for Mediating Pericyte-Endothelial Cell Interactions and Angiogenic Support in Combination Therapy of Human Umbilical Cord Perivascular Cells and Endothelial Progenitor Cells

Introduction: First Trimester Human Umbilical Cord Perivascular Cells (FTM HUCPVC) have higher cardiovascular regenerative potential compared to older sources of MSC. We designed a cell combination therapy with FTM HUCPVC and endothelial progenitor cells (EPC) to enhance angiogenesis after MI. However, the mechanism of PVC-EPC interaction is unclear. Hypothesis: Platelet derived growth factor beta receptor (PDGFB-R) expression of HUCPVC mediates homing and integration with endothelial networks and required for supporting neovasculature. Methods: Expression of pericyte markers PDGFB-R and CD146 of xeno-free expanded (HPL 2.5%) FTM and term HUCPVC was analyzed by flow cytometry. PDGFB-driven trans-ECM migration was assessed by Corning Biocoat Invasion assay with 1-100 ng/ml PDGFB. siRNA for PDGFB-R, CD146 and scrambled control was used with lipofectamine for 72 hours. Silencing was confirmed by qPCR and flow cytometry. 10% HPL was used for PDGFB-R desensitization. HUCPVC of all treatment groups were co-cultured with EPC and rat aortic rings on Matrigel TM . Cell migration, endothelial adhesion and network properties were quantified. Statistics: one-way ANOVA N=5 Results: PDGFB-R and CD146 expression and endothelial network augmenting function of FTM HUCPVC was significantly higher than of term HUCPVC. PDGFB dependent migration of FTM HUCPVC was disabled by both ligand (&lt;10%) and siRNA-based (&lt;5%) downregulation of PDGFB-R. Fluorescence microscopy (24 h) showed limited homing and endothelial interactions of PDGFB-R-reduced FTM HUCPVC. PDGFB-R-reduced FTM HUCPVC had significantly decreased network growth and nodes ( p &lt;0.0001) compared to control groups (day3). In EPC co-cultures, PDGFB-R-reduced FTM HUCPVC had limited endothelial interactions and coverage compared to control FTM HUCPVC. PDGFB-R-reduced FTM HUCPVC treated networks had significantly thinner ( p &lt;0.001) and shorter tubules ( p &lt;0.001) compared to control FTM HUCPVC groups. Down-regulation of C146 did not alter FTM HUCPVC pericyte properties. Conclusions: PDGFB-R is crucial for the homing and endothelial support of HUCPVC. This interaction is critical in vascular cell combination therapies and PDGFB-R expression can be a novel criterion for cardiovascular cell therapy.

In vitro study of human endothelial progenitor cells behaviour on nitrided Ni-free Ti–27Nb alloy

A superelastic Ni-free Ti–27Nb alloy has been synthesized and gas nitrided at high temperature to investigate its suitability for vascular implant applications. The cellular responses of human endothelial progenitor cells (EPCs) to both bare and nitrided Ti–27Nb alloy have been analyzed using Live/Dead staining, MTT assay, fluorescence microscopy and ELISA technique, as well as NiTi alloy for comparison. Live/Dead staining and MTT assay were performed to assess the cellular viability and proliferation, while fluorescence microscopy was used to analyze cell adhesion, cell morphology, and the expression of endothelial cell markers (VE-cadherin and von Willebrand factor). Secretion of the pro-inflammatory chemokine MCP (monocyte chemoattractant protein)-1 by the cells grown in contact with the analyzed materials was further verified using the enzyme-linked immunosorbent assay. The results obtained revealed that adhesion, spreading, viability, proliferation rate and phenotypic markers expression of EPCs were similar on the surfaces of Ti–27Nb and NiTi alloys. Cells exposed to nitrided Ti–27Nb surface exhibited significantly decreased inflammatory response, which may be beneficial for reducing in-stent restenosis incidence.

Isolating and expanding endothelial progenitor cells from peripheral blood on peptide-functionalized polystyrene surfaces.

The expansion of human peripheral blood endothelial progenitor cells to obtain therapeutically relevant endothelial colony-forming cells (ECFCs) has been commonly performed on xeno-derived extracellular matrix proteins. For cellular therapy applications, xeno-free culture conditions are desirable to improve product safety and reduce process variability. We have previously described a novel fluorophore-tagged RGD peptide (RGD-TAMRA) that enhanced the adhesion of mature endothelial cells in vitro. To investigate whether this peptide can replace animal-derived extracellular matrix proteins in the isolation and expansion of ECFCs, peripheral blood mononuclear cells from 22 healthy adult donors were seeded on RGD-TAMRA-modified polystyrene culture surfaces. Endothelial colony formation was significantly enhanced on RGD-TAMRA-modified surfaces compared to the unmodified control. No phenotypic differences were detected between ECFCs obtained on RGD-TAMRA compared to ECFCs obtained on rat-tail collagen-coated surfaces. Compared with collagen-coated surfaces and unmodified surfaces, RGD-TAMRA surfaces promoted ECFC adhesion, cell spreading, and clonal expansion. This study presents a platform that allows for a comprehensive in vitro evaluation of peptide-based biofunctionalization as a promising avenue for ex vivo ECFC expansion.

Isolation of induced pluripotent stem cell-derived endothelial progenitor cells from sac-like structures

Transplanted endothelial progenitor cells (EPCs) repair blood vessels and exert regenerative effects on disorders such as lower limb ischemia. EPCs serve as a model for pathophysiological and pharmacokinetic studies, which is important for drug discovery. However, primary human EPCs are phenotypically unstable, which limits their clinical utility. Therefore, we employed human induced pluripotent stem (iPS) cells to circumvent this problem. Here we focused on human iPS cell-derived sac-like structures (iPS-sacs), which contain endothelial lineage cells and hematopoietic lineage cells. Previous studies isolated only hematopoietic lineage cells from iPS-sacs. Therefore, here we attempted to isolate EPCs. However, iPS-sacs generated by a published protocol did not contain sufficient EPCs. Therefore, to generate iPS-sacs highly enriched in EPCs, we added the glycogen synthase kinase 3 beta (GSK3β) inhibitor CHIR-99021 to the culture medium early during differentiation. The cells rapidly differentiated into mesoderm to yield abundant EPCs, and CHIR-99021 increased the proportion of EPCs contained in iPS-sacs. EPCs, which were purified using anti-platelet endothelial cell adhesion molecule (PECAM1) antibody-conjugated beads, expressed markers of immature endothelial cells. Purified EPCs formed tube-like structures and incorporated acetylated low density lipoprotein (Ac-LDL), reflecting endothelial phenotypes. The simple method described here will likely improve regenerative medicine and facilitate basic studies on the endothelial lineage.

Normoxia is not favorable for maintaining stemness of human endothelial progenitor cells

The in vitro expansion of endothelial progenitor cells (EPCs) is necessary for obtaining sufficient amounts of cells for clinical applications. However, EPC expansion is conventionally carried out under non-physiologic oxygen concentrations (normoxia, ~20% O2). We compared the effects of normoxic and hypoxic culture on the stemness of expanded EPCs. Human EPCs were cultured under hypoxia (1% O2) or normoxia (~20% O2), respectively. Cell proliferation, colony formation, in vitro angiogenesis, and the migration ability of the expanded EPCs were compared. To explore the underlying mechanism, whole transcriptome RNA sequencing (RNA-seq) was also performed to select differentially expressed genes (DEGs), which were then partially validated by western blotting. EPCs cultured under normoxia showed reduced proliferation, colony formation, in vitro angiogenesis, and migration abilities and a higher proportion of senescent cells compared with those cultured under hypoxia. A total of 48 DEGs were identified by transcriptome RNA-seq. Further bioinformatics analysis revealed that six pathways were enriched, among which the p53 signaling pathway. Finally, we confirmed the differential expression of the p53 pathway by Western blot analysis. Compared with hypoxia, normoxia is not favorable for maintaining the stemness of human EPCs. Several signaling pathways, including the p53 signaling pathway implicate in reducing stemness of EPCs under normoxia.

A novel combination cell therapy using first trimester human umbilical cord-derived mesenchymal stromal cells and endothelial progenitor cells significantly improves cardiac recovery after myocardial infarction

Background & Aim Umbilical cord-derived MSC showed significant angiogenic potential via paracrine and direct cell-to-cell support to the vasculature. Endothelial progenitor cells (EPC) have the potential to develop vascular structures while maintaining paracrine properties following ischemic injury. Hypothesis A combination therapy of first trimester umbilical cord perivascular cells (FTM HUCPVC) with rat EPC will promote significant angiogenesis, cardiomyocyte survival and lead to functional cardiac recovery after MI. Methods, Results & Conclusion Methods MI was induced by LAD ligation on 8-week-old Foxrnu rats. 1 week later, rats with notable decrease ( Results 5-days after injection, exclusively G4 animals showed significant cardiac recovery, improved EF and FS compared to G1-G2-G3 (p Conclusions Our results indicate the superior potential of FTM HUCPVC - EPC combination cell therapy compared to single-cell-type treatments for myocardial regeneration and cardiac function recovery after MI.

Hydrogen sulfide improves vascular repair by promoting endothelial nitric oxide synthase-dependent mobilization of endothelial progenitor cells.

Endothelial progenitor cells (EPCs) play a crucial role in endothelial repair after arterial injury. Hydrogen sulfide (H2S) is a novel gasotransmitter that regulates vascular homeostasis. We investigated whether exogenous H2S could facilitate EPCs in repairing arterial injury. Sodium hydrosulfide (NaHS), a precursor of H2S, promoted re-endothelialization and inhibited neointima formation in a rodent carotid artery injury model. Flow cytometric analysis revealed that NaHS treatment significantly increased the yield of EPCs after vascular injury. Furthermore, NaHS enhanced the capacity of EPCs to the luminal surface of injured arteries in wild-type mice, which had received a bone marrow transplantation from tie2-GFP donor mice. However, this enhancing effect was greatly attenuated in endothelial nitric oxide synthase knockout mice (eNOS). In-vitro incubation of human EPCs with NaHS not only increased the yield of EPCs, but also enhanced their adhesion and colony formation capacities. Treatment with an eNOS inhibitor (L-NAME) blocked the effects of NaHS on EPCs functions. H2S enhances eNOS-dependent mobilization of bone marrow-derived EPCs and facilitates re-endothelialization following vascular injury.

Clinical Assessment of Intravenous Endothelial Progenitor Cell Transplantation in Dogs

Endothelial progenitor cells (EPCs) have been applied for cell therapy because of their roles in angiogenesis and neovascularization in ischemic tissue. However, adverse responses caused by EPC therapy have not been fully investigated. In this study, a human peripheral blood sample was collected from a healthy donor and peripheral blood mononuclear cells were separated using Ficoll-Hypaque. There were four experimental groups: 10 ml saline infusion group (injection rate; 3 ml/min), 10 ml saline bolus group (injection rate; 60 ml/min), 10 ml EPCs infusion group (2 x 105 cells/ml, injection rate; 3 ml/min), 10 ml EPCs bolus group (2 × 105 cells/ml, injection rate; 60 ml/min). Clinical assessment included physical examination and laboratory examination for intravenous human EPC transplantation in dogs. The results revealed no remarkable findings in vital signs among the dogs used. In blood analysis, platelet counts in saline infusion groups were significantly higher than in the EPC groups within normal ranges, and no significant differences were observed except K+, Cl– and blood urea nitrogen/urea. In ELISA assay, no significant difference was observed in serum tumor necrosis factor alpha. The serum concentration of vascular endothelial growth factor was significantly higher in EPC groups than in saline groups, and interleukin 10 was significantly up-regulated in the EPC infusion group compared with other groups. In conclusion, we demonstrated that no clinical abnormalities were detected after intravenous transplantation of human EPCs in dogs. The transplanted xenogenic EPCs might be involved in anti-inflammatory and angiogenic functions in dogs.

The Use of Live Cell Imaging and Automated Image Analysis to Assist With Determining Optimal Parameters for Angiogenic Assay in vitro.

Testing angiogenic potential and function of cells in culture is important for the understanding of the mechanisms that can modulate angiogenesis, especially when discovering novel anti- or pro-angiogenic therapeutics. Commonly used angiogenic assays include tube formation, proliferation, migration, and wound healing, and although well-characterized, it is important that methodology is standardized and reproducible. Human endothelial progenitor cells (EPCs) are critical for post-natal vascular homeostasis and can be isolated from human peripheral blood. Endothelial colony forming cells (ECFCs) are a subset of EPCs and are of interest as a possible therapeutic target for hypoxic diseases such as kidney disease, as they have a high angiogenic potential. However, once ECFCs are identified in culture, the exact timing of passaging has not been well-described and the optimal conditions to perform angiogenic assays such as seeding density, growth media (GM) concentrations and end-points of these assays is widely varied in the literature. Here, we describe the process of isolating, culturing and passaging ECFCs from patients with end-stage renal disease (ESRD), aided by image analysis. We further describe optimal conditions, for human bladder endothelial cells (hBECs), challenged in angiogenic assays and confirm that cell density is a limiting factor in accurately detecting angiogenic parameters. Furthermore, we show that GM along is enough to alter the angiogenic potential of cells, seeded at the same density. Lastly, we report on the success of human ECFCs in angiogenic assays and describe the benefits of live-cell imaging combined with time-lapse microscopy for this type of investigation.

Garcimultiflorone K inhibits angiogenesis through Akt/eNOS- and mTOR-dependent pathways in human endothelial progenitor cells

Background Garcimultiflorone K is a novel polyprenylated polycyclic acylphloroglucinol isolated from the stems of Garcinia multiflora that exhibits promising anti-angiogenic activity in human endothelial progenitor cells (EPCs). Purpose This study sought to determine the underlying anti-angiogenic mechanisms and pharmacological properties of garcimultiflorone K. Methods We examined the anti-angiogenic effects of garcimultiflorone K and its mechanisms of action using in vitro EPC models and in vivo zebrafish embryos. Results EPCs proliferation, migration, differentiation and capillary-like tube formation were effectively and concentration-dependently inhibited by garcimultiflorone K without any signs of cytotoxicity. Our investigations revealed that garcimultiflorone K suppressed EPCs angiogenesis through Akt, mTOR, p70S6K, and eNOS signaling cascades. Notably, garcimultiflorone K dose-dependently impeded angiogenesis in zebrafish embryos. Conclusion Our data demonstrate the anti-angiogneic effects of garcimultiflorone K in both in vitro and in vivo models. Garcimultiflorone K appears to have potential in the treatment of angiogenesis-related diseases.

The Role of Estrone in Endothelial Progenitor Cell Number and Function

Microvascular dysfunction is a key component of cardiovascular disease and estrogens, particularly estradiol, have been shown to be vascular protective. Endothelial progenitor cells (EPCs) are a stem cell population that supports the integrity of the microvasculature and show increased proliferation in response to estradiol exposure. The plasma concentration of EPCs negatively correlates with incidence of stroke and coronary artery disease. Another estrogen, estrone, has been understudied due to its weak binding at nuclear estrogen receptors ERa and ERb. However, recent evidence suggests that estrone is biologically active through a G protein coupled estrogen receptor, GPER. In some vascular cell populations, estrone independently increases proliferation and modulates the proliferative effects of estradiol. In pre‐menopausal women with high estradiol/estrone ratio, EPCs are present in sufficient numbers to enhance angiogenesis and repair vessels at sites of damage. After menopause when the estradiol/estrone ratio is low, EPC number is decreased, the microvasculature is compromised, and CVD risk increases. In the treatment of symptoms of menopause, we use a combination estrogen therapy which is largely estrone based. Using this therapy, estradiol levels are restored pharmacologically, but the estradiol/estrone ratio remains low. During treatment, EPC numbers remain low and cardiovascular risk remains high.Here we investigate the role of estrone in EPC proliferation via in vitro exposure studies using EPCs from human female blood samples and EPCs isolated from the bone marrow of Sprague Dawley Rats. We also assess how estrone, estradiol and combination exposures effect the ability of EPCs to enhance endothelial cell tube formation (vasculogenesis in vitro). Human and rat EPCs were defined as cells derived from the mononuclear cell fraction that after 10–14 days of expansion/selection in culture adhere to fibronectin, display typical endothelial cell (EC) morphology and are positive for the EPC markers VEGFR2, CD133, cKIT, CD34, and Dil‐ac‐LDL uptake. After sample collection (human peripheral blood or rat bone marrow), the mononuclear cell layer was isolated using density centrifugation. The mononuclear cell layer was then plated in endothelial growth media for 14 days. After 14 days the EPCs cells were used for expression, proliferation and tube formation assays. The proliferation assays were performed over a 24 hour exposure to 1 &amp; 2 nmol estrone, 1 &amp; 2 nmol estradiol and 1:1 (1nmol) estrone:estradiol. In our tube formation assay, EPCs were exposed to estrone, estradiol and 1:1 estrone:estradiol for 24 hours and then added to an endothelial cell tube formation assay. We assessed GPER expression via qPCR and immunocytochemistry on EPCs without hormone treatment.In vitro EPC proliferation was enhanced by both estrone and estradiol in a dose dependent manner. When estrone and estradiol were applied in a one to one ratio, resulting EPC proliferation was below that of control. GPER is expressed on both human and rat EPCs. Further studies will use a rat model to investigate physiological effects of estrone and GPER agonism on EPC ability to enhance angiogenesis in vivo.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Augmented Survival, Angiogenic Properties and Mobilization of Human Endothelial Progenitor Cells Following Exposure to Sevoflurane

Anesthetic preconditioning (APC) with volatile agents has cardiac anti‐ischemic virtues that may be linked to stimulating effects of volatile anesthetics on stem cells biology. We investigated the influence of sevoflurane (Sevo) on apoptosis, proliferation, incorporation in tube‐like structure and mobilization of human endothelial progenitor cells (EPCs). 7‐days old cultured EPCs derived from human cord blood mononuclear cells were exposed in vitro to sevoflurane 2% or 4% in air/5% CO2, or only to air/5% CO2 (sham control) for 2 hrs in a modular chamber. Proliferation, apoptosis and incorporation of EPCs into tube‐like structures of HUVEC was evaluated 24 hrs later. EPCs plasma levels were evaluated by flow‐cytometry and in vitro culture assay before exposure to sevoflurane (1 MAC in 50% O2) or only to 50% O2 (sham control) and 24 hrs afterwards, in blood samples collected from coronary artery disease patients. Values are expressed as means ± SEM, compared by ANOVA. In the in vitro preconditioning setting, sevoflurane stimulated EPCs proliferation estimated by the MTS assay (n = 12, p &lt; 0.01 for 2% Sevo, p &lt; 0.001 for 4% Sevo) and by counting adherent DilAcLDL+/ FITC‐UEAI+ cells (n = 4, p &lt; 0.01 for 2% Sevo, p &lt; 0.001 for 4% Sevo), diminished apoptosis measured by annexin V/ propidium iodide assay (n = 5, p &lt; 0.05 for both 2% and 4% Sevo), and raised the number of incorporated DiIAcLDL‐EPCs into HUVEC tube‐like structures (n = 4, p &lt; 0.01 for 2% Sevo, p &lt; 0.001 for 4% Sevo) when compared to control. Plasma CD45dim/CD34+/CD309+ and CD45dim/CD34+/CD309+/CD133+ mononuclear cell populations were increased in Sevo treated patients (n = 15) vs. the control group (n = 10) at 24 h post‐exposure vs. baseline (p &lt; 0.01), and the number of adherent DilAcLDL+/FITC‐UEAI+ cells in 7 days old EPCs cultures was significantly higher in the study group vs. control between the same time points (p &lt; 0.05). Our data prove that sevoflurane positively influences the biology of EPCs both in vivo and vitro, and may have an important cardioprotective impact in various interventional and surgical heart procedures by improving angiogenesis and ischemic tissue repair.Support or Funding InformationThe financial support was provided by UEFISCDI Romania, research grant PN‐II‐RUTE‐2012‐3‐0463.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Human Bone Marrow Endothelial Progenitor Cell Transplantation into Symptomatic ALS Mice Delays Disease Progression and Increases Motor Neuron Survival by Repairing Blood-Spinal Cord Barrier

Convincing evidence demonstrated impairment of the blood-spinal cord barrier (BSCB) in Amyotrophic Lateral Sclerosis (ALS), mainly by endothelial cell (EC) alterations. Replacing damaged ECs by cell transplantation is a potential barrier repair strategy. Recently, we showed that intravenous (iv) administration of human bone marrow CD34+ (hBM34+) cells into symptomatic ALS mice benefits BSCB restoration and postpones disease progression. However, delayed effect on motor function and some severely damaged capillaries were noted. We hypothesized that hematopoietic cells from a restricted lineage would be more effective. This study aimed to establish the effects of human bone marrow-derived endothelial progenitor cells (hBMEPCs) systemically transplanted into G93A mice at symptomatic disease stage. Results showed that transplanted hBMEPCs significantly improved behavioral disease outcomes, engrafted widely into capillaries of the gray/white matter spinal cord and brain motor cortex/brainstem, substantially restored capillary ultrastructure, significantly decreased EB extravasation into spinal cord parenchyma, meaningfully re-established perivascular astrocyte end-feet, and enhanced spinal cord motor neuron survival. These results provide novel evidence that transplantation of hBMEPCs effectively repairs the BSCB, potentially preventing entry of detrimental peripheral factors, including immune/inflammatory cells, which contribute to motor neuron dysfunction. Transplanting EC progenitor cells may be a promising strategy for barrier repair therapy in this disease.

Exosomes from endothelial progenitor cells improve outcomes of the lipopolysaccharide-induced acute lung injury

BackgroundThe acute respiratory distress syndrome (ARDS) is characterized by disruption of the alveolar-capillary barrier resulting in accumulation of proteinaceous edema and increased inflammatory cells in the alveolar space. We previously found that endothelial progenitor cell (EPC) exosomes prevent endothelial dysfunction and lung injury in sepsis in part due to their encapsulation of miRNA-126. However, the effects of EPC exosomes in acute lung injury (ALI) remain unknown.MethodsTo determine if EPC exosomes would have beneficial effects in ALI, intratracheal administration of lipopolysaccharide (LPS) was used to induce ALI in mice. Lung permeability, inflammation, and the role of miRNA-126 in the alveolar-epithelial barrier function were examined.ResultsThe intratracheal administration of EPC exosomes reduced lung injury following LPS-induced ALI at 24 and 48 h. Compared to placebo, intratracheal administration of EPC exosomes significantly reduced the cell number, protein concentration, and cytokines/chemokines in the bronchoalveolar lavage fluid (BALF), indicating a reduction in permeability and inflammation. Further, EPC exosomes reduced myeloperoxidase (MPO) activity, lung injury score, and pulmonary edema, demonstrating protection against lung injury. Murine fibroblast (NIH3T3) exosomes, which do not contain abundant miRNA-126, did not provide these beneficial effects. In human small airway epithelial cells (SAECs), we found that overexpression of miRNA-126-3p can target phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2), while overexpression of miRNA-126-5p inhibits the inflammatory alarmin HMGB1 and permeability factor VEGFα. Interestingly, both miR-126-3p and 5p increase the expression of tight junction proteins suggesting a potential mechanism by which miRNA-126 may mitigate LPS-induced lung injury.ConclusionsOur data demonstrated that human EPC exosomes are beneficial in LPS-induced ALI mice, in part through the delivery of miRNA-126 into the injured alveolus.

Effect of Polyethylene Terephthalate on Functional Properties of Endothelial and Mesenchymal Cells

We studied the influence of vascular prostheses made of polytetrafluoroethylene and polyethylene terephthalate on the proliferation, migration, and NO production by bone marrow mesenchymal stem cells, human endothelial progenitor cells, and EA.hy926 endothelial cells, colonization of the prosthesis surface by endothelial and mesenchymal cells was also analyzed. Synthetic prostheses have a negative effect on cell proliferation and migration, while surface treatment with proteins (fibronectin or gelatin) promotes colonization of the prostheses with cells.