Secretome of endothelial progenitor cells from stroke patients promotes endothelial barrier tightness and protects against hypoxia-induced vascular leakage

Abstract Background Cell-based therapeutic strategies have been proposed as an alternative for brain repair after stroke, but their clinical application has been hampered by potential adverse effects in the long term. The present study was designed to test the effect of the secretome of endothelial progenitor cells (EPCs) from stroke patients (scCM) on in vitro human models of angiogenesis and vascular barrier. Methods Two different scCM batches were analysed by mass spectrometry and a proteome profiler. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used for designing angiogenesis studies (proliferation, migration, and tubulogenesis) or in vitro models of EC monolayer (confluent monolayer ECs—CMECs) and blood–brain barrier (BBB; brain-like ECs—BLECs). Cells were treated with scCM (5 μg/mL) or protein-free endothelial basal medium (scEBM—control). CMECs or BLECs were exposed (6 h) to oxygen–glucose deprivation (OGD) conditions (1% oxygen and glucose-free medium) or normoxia (control—5% oxygen, 1 g/L of glucose) and treated with scCM or scEBM during reoxygenation (24 h). Results The analysis of different scCM batches showed a good reproducibility in terms of protein yield and composition. scCM increased CD34+-EC proliferation, tubulogenesis, and migration compared to the control (scEBM). The proteomic analysis of scCM revealed the presence of growth factors and molecules modulating cell metabolism and inflammatory pathways. Further, scCM decreased the permeability of CMECs and upregulated the expression of the junctional proteins such as occludin, VE-cadherin, and ZO-1. Such effects were possibly mediated through the activation of the interferon pathway and a moderate downregulation of Wnt signalling. Furthermore, OGD increased the permeability of both CMECs and BLECs, while scCM prevented the OGD-induced vascular leakage in both models. These effects were possibly mediated through the upregulation of junctional proteins and the regulation of MAPK/VEGFR2 activity. Conclusion Our results suggest that scCM promotes angiogenesis and the maturation of newly formed vessels while restoring the BBB function in ischemic conditions. In conclusion, our results highlight the possibility of using EPC-secretome as a therapeutic alternative to promote brain angiogenesis and protect from ischemia-induced vascular leakage.

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Gene transfer of endothelial NO-synthase restores migratory capacity of endothelial progenitor cells from patients with coronary artery diseases

Clinical & Investigative Medicine ◽

10.25011/cim.v30i4.2877 ◽

2007 ◽

Vol 30 (4) ◽

pp. 96

Author(s):

Michael R. Ward ◽

Qiuwang Zhang ◽

Duncan J. Stewart ◽

Michael J.B. Kutryk

Keyword(s):

Risk Factors ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Fold Increase ◽

No Synthase ◽

Endothelial Progenitor ◽

Migratory Capacity ◽

Acute Mi ◽

Cad Risk

Autologous endothelial progenitor cells (EPCs) have been used extensively in the development of cell-based therapy for acute MI. However, EPCs isolated from patients with CAD and/or CAD risk factors have reduced regenerative activity compared to cells from healthy subjects. As in endothelial cells, endothelial NO synthase (eNOS) expression and subsequent NO production are believed to be critical determinants of EPC function. Recently, the ability of EPCs to migrate in vitro in response to chemotactic stimuli has been shown to predict their regenerative capacity in clinical studies. Therefore, we hypothesized that the regenerative function of EPCs from patients with or at high risk for CAD will be enhanced by overexpression of eNOS, as assessed by migratory capacity. Methods: EPCs were isolated from the blood of human subjects with CAD risk factors (>15% Framingham risk score; FRS) (± CAD) by Ficoll gradient separation and differential culture. Following 3 days in culture, cells were transduced using lentivirus vectors containing either eNOS or GFP (sham) at an MOI of 3. The cells were cultured for an additional 5 days before being used in functional assays. Cell migration and chemotaxis in response to VEGF (50 ng/mL) and SDF-1 (100 ng/mL) were assessed using a modified Boyden Chamber assay. Results: Transduction at an MOI of 3 led to a ~90-100-fold increase in eNOS mRNA expression and a 5-6 fold increase in eNOS protein expression, as assessed by qRT-PCR and Western Blotting. Moreover, there was a significant improvement in the migration of EPCs following eNOS transduction compared to sham-transduced EPCs in response to both VEGF (44.3 ± 8.4 vs. 31.1 ± 4.6 cells/high power field; n=10, p < 0.05) and SDF-1 (51.9 ± 11.1 vs. 34.5 ± 3.3 cells/HPF; n=10, p < 0.05). Conclusions: These data show that the reduced migration capacity of EPCs isolated from patients with CAD and/or CAD risk factors can be significantly improved through eNOS overexpression in these cells. Thus, eNOS transduction of autologous EPCs may enhance their ability to restore myocardial perfusion and function following acute MI. We intend to further explore the regenerative potential of eNOS-transduced EPCs using various in vitro and in vivo models.

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Effective Actions of Ion Release from Mesoporous Bioactive Glass and Macrophage Mediators on the Differentiation of Osteoprogenitor and Endothelial Progenitor Cells

Pharmaceutics ◽

10.3390/pharmaceutics13081152 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1152

Author(s):

Alberto Polo-Montalvo ◽

Laura Casarrubios ◽

María Concepción Serrano ◽

Adrián Sanvicente ◽

María José Feito ◽

...

Keyword(s):

Bone Regeneration ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Mesoporous Structure ◽

Ion Release ◽

Endothelial Progenitor ◽

Matrix Mineralization ◽

Intracellular Content

Due to their specific mesoporous structure and large surface area, mesoporous bioactive glasses (MBGs) possess both drug-delivery ability and effective ionic release to promote bone regeneration by stimulating osteogenesis and angiogenesis. Macrophages secrete mediators that can affect both processes, depending on their phenotype. In this work, the action of ion release from MBG-75S, with a molar composition of 75SiO2-20CaO-5P2O5, on osteogenesis and angiogenesis and the modulatory role of macrophages have been assessed in vitro with MC3T3-E1 pre-osteoblasts and endothelial progenitor cells (EPCs) in monoculture and in coculture with RAW 264.7 macrophages. Ca2+, phosphorous, and silicon ions released from MBG-75S were measured in the culture medium during both differentiation processes. Alkaline phosphatase activity and matrix mineralization were quantified as the key markers of osteogenic differentiation in MC3T3-E1 cells. The expression of CD31, CD34, VEGFR2, eNOS, and vWF was evaluated to characterize the EPC differentiation into mature endothelial cells. Other cellular parameters analyzed included the cell size and complexity, intracellular calcium, and intracellular content of the reactive oxygen species. The results obtained indicate that the ions released by MBG-75S promote osteogenesis and angiogenesis in vitro, evidencing a macrophage inhibitory role in these processes and demonstrating the high potential of MBG-75S for the preparation of implants for bone regeneration.

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Effect and Mechanism of Thrombospondin-1 on the Angiogenesis Potential in Human Endothelial Progenitor Cells: An In Vitro Study

PLoS ONE ◽

10.1371/journal.pone.0088213 ◽

2014 ◽

Vol 9 (2) ◽

pp. e88213 ◽

Cited By ~ 13

Author(s):

Qing Qin ◽

Juying Qian ◽

Lei Ge ◽

Li Shen ◽

Jianguo Jia ◽

...

Keyword(s):

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

In Vitro Study ◽

Vitro Study ◽

Endothelial Progenitor ◽

Thrombospondin 1

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The role of eNOS in the migration and proliferation of bone-marrow derived endothelial progenitor cells and in vitro angiogenesis

Cell Biology International ◽

10.1002/cbin.10405 ◽

2015 ◽

Vol 39 (4) ◽

pp. 484-490 ◽

Cited By ~ 17

Author(s):

Aizhen Lu ◽

Libo Wang ◽

Liling Qian

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Endothelial Progenitor ◽

In Vitro Angiogenesis

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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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Effect of co-culture human endothelial progenitor cells with porcine oocytes during maturation and subsequent embryo development of parthenotes in vitro

Molecular Reproduction and Development ◽

10.1002/mrd.22969 ◽

2018 ◽

Vol 85 (4) ◽

pp. 336-347 ◽

Cited By ~ 3

Author(s):

Seok Hee Lee ◽

Hyun Ju Oh ◽

Min Jung Kim ◽

Erif M.N. Setyawan ◽

Yoo Bin Choi ◽

...

Keyword(s):

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Embryo Development ◽

Endothelial Progenitor

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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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