Nrf2 deficiency diminishes angiogenic potential of endothelial progenitor cells in vitro but improves neovascularization under ischemic conditions in vivo

Background: Endothelial progenitor cells (EPCs) have been shown to have angiogenic potential contributing to neovascularization. However, the definition of EPC, including surface marker expression of EPCs promoting vasculo-/angiogenesis in ischemic tissue, remains uncertain. We hypothesized that stem/progenitor (c-kit vs. sca-1) and endothelial cell (EC) markers (CD31) may identify cells with enhanced EPC potential. Methods and Results: Mononuclear cells (MNCs) were isolated from mouse bones, and Lin+ cells were depleted by magnetic cell sorting. Lin- cells were further sorted with the following markers (% of total MNCs) by FACS: c-kit+ (1.87%), sca-1+ (0.6%), c-kit+ /CD31+ (1.1%) and sca-1+ /CD31+ (0.28%). Non-sorted MNCs were used as a control. To examine EC phenotype in culture, cells were labeled with DiI and co-cultured with mature ECs (human microvascular endothelial cells: HMVECs). The percent incorporation of DiI labeled cells into HMVEC tube structures 12 hours after co-culture and BS1-lectin positivity/acLDL uptake were: sca-1+ /CD31+ cells (87 ± 2%) > c-kit+ /CD31+ (79 ± 8%) > sca-1+ (62 ± 8%) > c-kit+ (59 ± 5%) > MNC (50 ± 3% ) . Next, we examined homing capacity of these cells to ischemic myocardium using a mouse myocardial infarction (MI) model. DiI-labeled cells (5x10 4 , IV) were injected to splenectomized mice 3 days after MI, and the hearts were excised 24 hours after the cell injection for histological analysis. Interestingly, the number of recruited/retained DiI-labeled-cells in the MI hearts exactly replicated the findings of the in vitro tube formation assay (cells/HPF): sca-1+ /CD31+ (108 ± 26) > c-kit+ /CD31+ (77 ± 16) > sca-1+ (71 ± 14) > c-kit+ (67 ± 1) > MNCs (48 ± 6) , suggesting that sca-1+ /CD31+ cells might have great functional activities as endothelial precursors. Conclusions: Both stem/progenitor marker Sca-1 and EC marker CD31 expressing EPCs exhibited high potential angiogenic capacity with EC phenotypic features compared with c-kit expressing cells. Our data suggest that Sca-1+ /CD31+ cells may represent EPC-rich cell population, and Sca-1/CD31 could be useful markers to enrich for cells with EPC potential. Ongoing studies will determine the in vivo characteristics of these cells for ischemic tissue repair.

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Interleukin-1β Augments Angiogenic Responses of Murine Endothelial Progenitor Cells in Vitro

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2009.17 ◽

2009 ◽

Vol 29 (5) ◽

pp. 933-943 ◽

Cited By ~ 48

Author(s):

Anna Rosell ◽

Ken Arai ◽

Josephine Lok ◽

Tongrong He ◽

Shuzhen Guo ◽

...

Keyword(s):

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Mononuclear Cells ◽

Therapeutic Angiogenesis ◽

Interleukin 1Β ◽

Endothelial Progenitor ◽

Angiogenic Potential ◽

Diphenyl Tetrazolium Bromide ◽

In Vitro Angiogenesis

Endothelial progenitor cells (EPCs) may provide novel opportunities for therapeutic angiogenesis after ischemic diseases. However, it is unclear how the angiogenic potential of EPCs might be affected by an inflammatory environment. We examine how the potent cytokine interleukin-1β (IL-1β) affects angiovasculogenic responses in EPCs in culture. Mononuclear cells isolated from mouse spleen were plated on fibronectin-coated wells and grown in EGM-2 MV media. Endothelial progenitor cells were phenotyped using multiple markers (UEA-Lectin, ac-LDL, CD133, CD34, vWillebrand Factor, Flk-1) and to identify the IL-1 Receptor-I. We quantified cell and colony counts and performed MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide) and Matrigel assays, in vitro, under control and IL-1β (10 ng/mL) conditions. Endothelial progenitor cells exposed to IL-1β increased in the number of cells and colonies compared with untreated cells, without any effect on cell metabolic integrity. Furthermore, IL-1β treatment augmented EPC angiogenic function, significantly increasing the number of vessel-like structures in the Matrigel assay. An early phosphorylation of ERK1/2 occurred after IL-1β stimulation, and this pathway was inhibited if IL-1 Receptor-I was blocked. Our results suggest that IL-1β is a potent stimulator of in vitro angiogenesis through ERK signaling in mouse EPCs. Further studies are warranted to assess how interactions between proinflammatory environments and EPC responses may be leveraged to enhance therapeutic angiogenesis.

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Abstract 3955: Chemokines Fused to a Fractalkine Backbone and a GPI-Anchor Attract Embryonic Endothelial Progenitor Cells to the Site of Ischemia

Circulation ◽

10.1161/circ.118.suppl_18.s_509-a ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Chiraz El-Aouni ◽

Franziska Globisch ◽

Achim Pfosser ◽

Georg Stachel ◽

Rabea Hinkel ◽

...

Keyword(s):

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Adhesion Molecule ◽

Capillary Density ◽

Gpi Anchor ◽

Endothelial Progenitor ◽

Collateral Growth ◽

Artificial Adhesion

Recruitment of endothelial progenitor cells to the sites of ischemia is a prerequisite for efficient therapeutic neovascularization via vasculogenesis. Chemokines play a major role in the homing of EPCs at the ischemic vasculature, a mechanism fading in chronic ischemia. To overcome this limitation, we constructed an artificial adhesion molecule consisting of a GPI-anchor, a fractalkine-backbone and an SDF-1 head (SDF-1-fra-GPI), which was applied for enhanced recruitment of embryonic EPCs (eEPCs: CXCR4++, Tie2++, Thrombomodulin++, CD34-, MHCI-, vWF inducible, eNOS inducible) in vitro and in vivo . Methods: In a flow chamber adhesion assay, Control plasmids (pcDNA or GPI-SDF-1 cDNA) were compared to the SDF-1-fra-GPI construct for eEPC recruitment 24h after liposomal transfection of rat endothelial cells. In vivo, in rabbits (n=5 per group) at day 7 (d7) after femoral artery excision, 1 mg of the SDF-1-fra-GPI or eGFP cDNA was transfected into the ischemic limb. At d9, ischemic hindlimbs were retroinfused with 5x10 6 eEPCs. Angiography was performed for collateral quantification and frame count score at d9 and d37 (% of d9), capillary density was assessed via PECAM-1-staining (capillaries/muscle fiber = c/mf). Results: In vitro, eEPC adhesion (16±12 cells/field) was increased to a higher extent by SDF-1-fra-GPI (79±13) than SDF1-GPI (54±8) or control vector (37±8). In vivo , eEPC adhesion in the ischemic hindlimb after SDF-1-fra-GPI transfection compared to mock transfection (30±3 vs. 9±1 cells/field). Whereas capillary density was unaffected (1.66±0.30 SDF-1-Fra-GPI vs. 1.56±0.29 eEPCs), collateral growth (152±10% SDF-1-fra-GPI vs. 124±13%) as well as perfusion score (198±17% SDF-1-fra-GPI vs.160±6% eEPCs) further increased after SDF-1-fra-GPI transfection (controls: 1.24±0.12 c/mf, collaterals 105±8%, perfusion score 112±11%). We conclude that recruitment of EPCs expressing CXCR4 (the SDF-1 receptor) may benefit from pre-treatment of the recipient vasculature with SDF-1-Fra-GPI, an artificial adhesion molecule. This approach might be valuable for enhancing EPC recruitment in the scenario of therapeutic neovascular-ization of chronic ischemic syndromes.

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Characterization of Angiogenic Potential of Late Endothelial Progenitor Cells on Human Cord Blood and Bone Marrow

Blood ◽

10.1182/blood.v112.11.5404.5404 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5404-5404

Author(s):

Eun-Sun Yoo ◽

Jee-Young Ahn ◽

KiHwan Kwon ◽

Soo-Ah Oh ◽

Moon-Young Choi ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Adhesion Molecule ◽

Mononuclear Cells ◽

Marker Genes ◽

Endothelial Progenitor ◽

Angiogenic Potential ◽

Cell Based Therapy

Abstract Background: The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. Recently, we have demonstrated that there are 2 distinct types of EPCs from UCB having different biologic properties for angiogenic capabilities in vitro and in vivo. In present study, the aim is to directly compare umbilical cord blood (UCB)- and BM-derived late EPC surface phenotypes and in vitro functional capacity. Methods: Mononuclear cells from UCB and BM cultured using EGM-2 medium with VEGF, IGF-1 and FGF for 21 days. Late outgrowing endothelail cells(late OECs) which were in peak growth at third weeks of culture were analyzed for expression of various surface markers by flow cytometry/RT-PCR/IF, tube formation in Matrigel plates, proliferation assay, endothelial colony assay and the role of SDF-1/VEGF on migration. Results: The adherent cells after culture of 7 days exhibited a fibroblast like shape in BM and a cobblestone shaped cells in UCB. Although two sources of OECs were comparable in expression of endothelial and various adhesion molecule markers, BM-derived OECs contained higher proportion of cells expressing smooth muscle cell markers(SMMHC), several adhesion molecule(CD49d, CD62L and VCAM-1), whereas the expression of CXCR-4, PECAM-1 and CD62E and expression of mRNA on endothelial marker genes were higher in UCB-derived OECs. UCB-OECS stained positive for uptake of acetylated low-density lipoprotein and had more migratory ability in the presence of SDF-1 and VEGF compared with BM-OECs. Both sources OECs effectively formed capillary tubes in Matrigel plates. Conclusion: We directly compared OECs derived from UCB and BM and two source of OECs differ in aspect of several adhesion molecule and angiogenic potential in vitro. These difference of UCB render it potentially advantageous for human therapeutic OECs applications for potential applications for a “cell therapy” in the situations on vascular injuries when compared with patients-derived BM.

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Effects of androgens on endothelial progenitor cells in vitro and in vivo

Clinical Science ◽

10.1042/cs20090077 ◽

2009 ◽

Vol 117 (10) ◽

pp. 355-364 ◽

Cited By ~ 22

Author(s):

Gian Paolo Fadini ◽

Mattia Albiero ◽

Andrea Cignarella ◽

Chiara Bolego ◽

Christian Pinna ◽

...

Keyword(s):

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Endothelial Progenitor Cell ◽

Cell Biology ◽

Progenitor Cell ◽

Adult Males ◽

Endothelial Progenitor ◽

Healthy Human

The beneficial or detrimental effects of androgens on the cardiovascular system are debated. Endothelial progenitor cells are bone-marrow-derived cells involved in endothelial healing and angiogenesis, which promote cardiovascular health. Oestrogens are potent stimulators of endothelial progenitor cells, and previous findings have indicated that androgens may improve the biology of these cells as well. In the present study, we show that testosterone and its active metabolite dihydrotestosterone exert no effects on the expansion and function of late endothelial progenitors isolated from the peripheral blood of healthy human adult males, whereas they positively modulate early ‘monocytic’ endothelial progenitor cells. In parallel, we show that castration in rats is followed by a decrease in circulating endothelial progenitor cells, but that testosterone and dihydrotestosterone replacement fails to restore endothelial progenitor cells towards normal levels. This is associated with persistently low oestrogen levels after androgen replacement in castrated rats. In a sample of 62 healthy middle-aged men, we show that circulating endothelial progenitor cell levels are more directly associated with oestradiol, rather than with testosterone, concentrations. In conclusion, our results collectively demonstrate that androgens exert no direct effects on endothelial progenitor cell biology in vitro and in vivo.

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Humanized large-scale expanded endothelial colony–forming cells function in vitro and in vivo

Blood ◽

10.1182/blood-2008-09-181362 ◽

2009 ◽

Vol 113 (26) ◽

pp. 6716-6725 ◽

Cited By ~ 138

Author(s):

Andreas Reinisch ◽

Nicole A. Hofmann ◽

Anna C. Obenauf ◽

Karl Kashofer ◽

Eva Rohde ◽

...

Keyword(s):

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Large Scale ◽

Proliferative Potential ◽

Vascular Networks ◽

Endothelial Progenitor ◽

Endothelial Colony Forming Cells ◽

Promising Tool

Abstract Endothelial progenitor cells are critically involved in essential biologic processes, such as vascular homeostasis, regeneration, and tumor angiogenesis. Endothelial colony–forming cells (ECFCs) are endothelial progenitor cells with robust proliferative potential. Their profound vessel-forming capacity makes them a promising tool for innovative experimental, diagnostic, and therapeutic strategies. Efficient and safe methods for their isolation and expansion are presently lacking. Based on the previously established efficacy of animal serum–free large-scale clinical-grade propagation of mesenchymal stromal cells, we hypothesized that endothelial lineage cells may also be propagated efficiently following a comparable strategy. Here we demonstrate that human ECFCs can be recovered directly from unmanipulated whole blood. A novel large-scale animal protein-free humanized expansion strategy preserves the progenitor hierarchy with sustained proliferation potential of more than 30 population doublings. By applying large-scale propagated ECFCs in various test systems, we observed vascular networks in vitro and perfused vessels in vivo. After large-scale expansion and cryopreservation phenotype, function, proliferation, and genomic stability were maintained. For the first time, proliferative, functional, and storable ECFCs propagated under humanized conditions can be explored in terms of their therapeutic applicability and risk profile.

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Low Ethanol Concentrations Promote Endothelial Progenitor Cell Capacity and Reparative Function

Cardiovascular Therapeutics ◽

10.1155/2020/4018478 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Lars Brodowski ◽

Bianca Schröder-Heurich ◽

Berina Kipke ◽

Cara Schmidt ◽

Constantin S. von Kaisenberg ◽

...

Keyword(s):

Cell Surface ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Cell Interaction ◽

Cell Number ◽

Endothelial Progenitor ◽

Tubule Formation ◽

Close Link

Background. Endothelial progenitor cells (EPCs) are recruited to injured endothelium and contribute to its regeneration. There is evidence that moderate ethanol consumption prevents the development and progression of atherosclerosis in a variety of in vitro and in vivo models and increases the mobilization of progenitor cells. Furthermore, there are studies that identified ethanol at low concentration as a therapeutic tool to mobilize progenitor cells in peripheral blood. At the same time, the cell number of EPCs represents a close link to cardiovascular system constitution and function and contributes to cardiovascular risk. The aim of this study was to evaluate the effect of low dose ethanol on typical features of endothelial colony-forming cells (ECFCs), a proliferative subtype of EPCs. Methods and Results. We tested whether ethanol impacts the functional abilities of ECFC (e.g., migration, tube formation, and proliferation) using in vitro assays, the intercommunication of ECFC by exploring cell surface molecules by flow cytometry, and the expression of (anti-)angiogenic molecules by ELISA. Low concentrations of ethanol concentration promoted migration, proliferation, and tubule formation of ECFC. The expression of the cell surface marker VE-cadherin, a protein which plays an important role in cell-cell interaction, was enhanced by ethanol, while (anti-)angiogenic molecule expression was not impacted. Conclusion. Ethanol at moderate concentrations increases the angiogenic abilities of endothelial progenitor cells thus possibly contributing to vasoprotection.

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Role of β2-integrins for homing and neovascularization capacity of endothelial progenitor cells

Journal of Experimental Medicine ◽

10.1084/jem.20041402 ◽

2004 ◽

Vol 201 (1) ◽

pp. 63-72 ◽

Cited By ~ 230

Author(s):

Emmanouil Chavakis ◽

Alexandra Aicher ◽

Christopher Heeschen ◽

Ken-ichiro Sasaki ◽

Ralf Kaiser ◽

...

Keyword(s):

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Ex Vivo ◽

Limb Ischemia ◽

Endothelial Progenitor ◽

Cell Monolayers ◽

Β2 Integrins

The mechanisms of homing of endothelial progenitor cells (EPCs) to sites of ischemia are unclear. Here, we demonstrate that ex vivo–expanded EPCs as well as murine hematopoietic Sca-1+/Lin− progenitor cells express β2-integrins, which mediate the adhesion of EPCs to endothelial cell monolayers and their chemokine-induced transendothelial migration in vitro. In a murine model of hind limb ischemia, Sca-1+/Lin− hematopoietic progenitor cells from β2-integrin–deficient mice are less capable of homing to sites of ischemia and of improving neovascularization. Preactivation of the β2-integrins expressed on EPCs by activating antibodies augments the EPC-induced neovascularization in vivo. These results provide evidence for a novel function of β2-integrins in postnatal vasculogenesis.

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Abstract 894: Leptin Potentiates the Angiogenic Properties of Endothelial Progenitor Cells In Vitro and In Vivo

Circulation ◽

10.1161/circ.116.suppl_16.ii_175-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Nana-Maria Heida ◽

Marco R Schroeter ◽

I-Fen Cheng ◽

Elena I Deryugina ◽

Thomas Korff ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Tube Length ◽

Endothelial Progenitor ◽

Signal Transduction Inhibitors ◽

Stimulation Of

Endothelial progenitor cells (EPC) have been reported to contribute to neovascularization. We have previously shown that the adipocytokine leptin may enhance the adhesive properties of EPC by upregulating specific integrins. To investigate whether the angiogenic effects of leptin may be mediated by modulation of EPC function, mononuclear cells were isolated from healthy human volunteers and cultivated under endothelial cell conditions for 7 days. In the matrigel assay, pretreatment of EPC with recombinant leptin for 24 hours dose-dependently enhanced their incorporation into tubular structures provided by mature endothelial cells. For example, 138.3 ± 7.6% (P = 0.001) and 145.3 ± 5.5% (P = 0.0001) CM-DiI-labeled EPC were detected after stimulation with 10 and 100 ng/mL leptin, respectively (control-treated EPC defined as 100%). Furthermore, in the spheroid angiogenesis assay, stimulation of EPC with 10 ng/mL leptin increased the number of sprouts (P < 0.0001) and tube length (P < 0.0001) of coincubated mature endothelial cells, and the outgrowth of EPC (P < 0.0001). Addition of 100-fold excess of leptin-neutralizing or leptin-receptor-binding antibodies completely reversed these effects. Moreover, EPC adhesion onto endothelial cell tubules could be reduced by addition of RGD peptides (from 159 ± 13.7% to 101.8 ± 14.6%; P = 0.02), or of neutralizing antibodies against αvβ3 (from 165.3 ± 11.8% to 103.8 ± 13.3%; P = 0.006) or αvβ5 (to 93.5 ± 15.8%; P = 0.005). Further experiments using specific signal transduction inhibitors (10 μM of LY294002, PD98059, or SB203580), as well as Western blot analysis, revealed that leptin signaling in EPC involves phosphoinositide-3 kinase and p42/44, but not by p38 MAP kinase. The effects of leptin could also be confirmed under in vivo conditions. Stimulation of EPC with 100 ng/mL leptin potentiated the insprout of newly formed avian vessels into collagen onplants placed on the chorion allantoic membrane of chicken embryos (angiogenic index, 0.58 ± 0.24) compared to control-treated EPC (0.44 ± 0.27; P = 0.07) and endothelial basal medium alone (0.31 ± 0.26; P = 0.0007). Thus, our in vitro and in vivo results suggest that the angiogenic effects of leptin may partly depend on its specific interaction with endothelial progenitor cells.

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Hydrogen Sulfide-Preconditioning of Human Endothelial Progenitor Cells Transplantation Improves Re-Endothelialization in Nude Mice with Carotid Artery Injury

Cellular Physiology and Biochemistry ◽

10.1159/000480411 ◽

2017 ◽

Vol 43 (1) ◽

pp. 308-319 ◽

Cited By ~ 4

Author(s):

Xiao Ke ◽

Jun Zou ◽

Qingsong Hu ◽

Xiaoqing Wang ◽

Chengheng Hu ◽

...

Keyword(s):

Carotid Artery ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Nude Mice ◽

No Production ◽

Carotid Artery Injury ◽

Endothelial Progenitor ◽

Artery Injury

Background/Aims: The aim of present study was to test the hypothesis that preconditioning with sodium hydrosulfide (NaHS) could enhance the capacity of migration, adhesion and proliferation of endothelial progenitor cells (EPCs) in vitro, and also could improve the efficacy of EPCs transplantation for re-endothelialization in nude mice with carotid artery injury. The paper further addressed the underlying mechanisms. Methods: EPCs were isolated from peripheral blood mononuclear cells of healthy male volunteers and the markers of EPCs were analyzed by flow cytometry. Thereafter, different concentrations of NaHS (25, 50, 100, 200 and 500 uM) were used for preconditioning EPCs. In vitro and in vivo migration, adhesion and proliferation as well as nitric oxide (NO) production of EPCs were evaluated. Carotid artery injury model was produced in nude mice and thereafter, NaHS-preconditioned EPCs were transplanted in order to evaluate their capacity of re-endothelialization. Results: Cellular immuno-staining showed that isolated cells expressed the key markers of EPCs. In vitro, EPCs proliferation rates and NO production were gradually increased in a NaHS-concentration dependent manner, while these benefits were blocked at a concentration of 500 uM NaHS. Similarly, the migration and adhesion rates of EPCs were also increased the most prominently at a concentration of 200 µM NaHS. In vivo, compared to the control group, treatment with NaHS-preconditioned EPCs significantly enhanced the capacity of re-endothelialization of EPCs. Fluorescent microscope revealed that there were more EPCs homing to the injury vessels in the NaHS-preconditioned EPCs group than the non-preconditioned group. With the administration of AMPK or eNOS inhibitors respectively, the above benefits of NaHS-preconditioning were abrogated. Conclusion: These results suggested that NaHS-preconditioning enhanced the biological function and re-endothelialization of EPCs through the AMPK/eNOS signaling pathway.

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