Gleevec and Rapamycin Synergistically Reduce Cell Viability and Inhibit Proliferation and Angiogenic Function of Mouse Bone Marrow-Derived Endothelial Progenitor Cells

Objective: This study investigates the synergistic effects of Gleevec (imatinib) and rapamycin on the proliferative and angiogenic properties of mouse bone marrow-derived endothelial progenitor cells (EPCs). Materials and Methods: EPCs were isolated from mouse bone marrow and treated with different concentrations of Gleevec or rapamycin individually or in combination. The cell viability and proliferation were examined using the MTT assay. An analysis of cell cycle and apoptosis was performed using flow cytometry. Formation of capillary-like tubes was examined in vitro, and the protein expression of cell differentiation markers was determined using Western blot analysis. Results: Gleevec significantly reduced cell viability, cell proliferation, and induced cell apoptosis in EPCs. Rapamycin had similar effects on EPCs, but it did not induce cell apoptosis. The combination of Gleevec and rapamycin reduced the cell proliferation but increased cell apoptosis. Although rapamycin had no demonstratable effect on tube formation, the combined therapy of Gleevec and rapamycin significantly reduced tube formation when compared with Gleevec alone. Mechanistically, Gleevec, but not rapamycin, induced a significant elevation in caspase-3 activity in EPCs, and it attenuated the expression of the endothelial protein marker platelet-derived growth factor receptor α. Functionally, rapamycin, but not Gleevec, significantly enhanced the expression of endothelial differentiation marker proteins, while attenuating the expression of mammalian target of rapamycin signaling-related proteins. Conclusions: Gleevec and rapamycin synergistically suppress cell proliferation and tube formation of EPCs by inducing cell apoptosis and endothelial differentiation. Mechanistically, it is likely that rapamycin enhances the proapoptotic and antiangiogenic effects of Gleevec by promoting the endothelial differentiation of EPCs. Given that EPCs are involved in the pathogenesis of some cardiovascular diseases and critical to angiogenesis, pharmacological inhibition of EPC proliferation by combined Gleevec and rapamycin therapy may be a promising approach for suppressing cardiovascular disease pathologies associated with angiogenesis.

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Efficient nano iron particle-labeling and noninvasive MR imaging of mouse bone marrow-derived endothelial progenitor cells

International Journal of Nanomedicine ◽

10.2147/ijn.s16934 ◽

2011 ◽

pp. 511 ◽

Cited By ~ 18

Author(s):

Chen ◽

Jia

Keyword(s):

Bone Marrow ◽

Mr Imaging ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Iron Particle ◽

Mouse Bone Marrow ◽

Endothelial Progenitor

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Vascular endothelial growth factor-A signaling in bone marrow-derived endothelial progenitor cells exposed to hypoxic stress

Physiological Genomics ◽

10.1152/physiolgenomics.00070.2013 ◽

2013 ◽

Vol 45 (21) ◽

pp. 1021-1034 ◽

Cited By ~ 22

Author(s):

Brian R. Hoffmann ◽

Jordan R. Wagner ◽

Anthony R. Prisco ◽

Agnieszka Janiak ◽

Andrew S. Greene

Keyword(s):

Bone Marrow ◽

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Signaling Pathways ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Tube Formation ◽

Vascular Endothelial ◽

Endothelial Progenitor ◽

Endothelial Growth Factor

Bone marrow-derived endothelial progenitor cells (BM-EPCs) are stimulated by vascular endothelial growth factor-A (VEGF-A) and other potent proangiogenic factors. During angiogenesis, an increase in VEGF-A expression stimulates BM-EPCs to enhance endothelial tube formation and contribute to an increase in microvessel density. Hypoxia is known to produce an enhanced angiogenic response and heightened levels of VEGF-A have been seen in oxygen deprived epithelial and endothelial cells, yet the pathways for VEGF-A signaling in BM-EPCs have not been described. This study explores the influence of hypoxia on VEGF-A signaling in rat BM-EPCs utilizing a novel proteomic strategy to directly identify interacting downstream components of the combined VEGF receptor(s) signaling pathways, gene expression analysis, and functional phenotyping. VEGF-A signaling network analysis following liquid chromatographic separation and tandem mass spectrometry revealed proteins related to inositol/calcium signaling, nitric oxide signaling, cell survival, cell migration, and inflammatory responses. Alterations in BM-EPC expression of common angiogenic genes and tube formation in response to VEGF-A during hypoxia were measured and combined with the proteomic analysis to enhance and support the signaling pathways detected. BM-EPC tube formation assays in response to VEGF-A exhibited little tube formation; however, a cell projection/migratory phenotype supported the signaling data. Additionally, a novel assay measuring BM-EPC incorporation into preformed endothelial cell tubes indicated a significant increase of incorporated BM-EPCs after pretreatment with VEGF-A during hypoxia. This study verifies known VEGF-A pathway components and reveals several unidentified mechanisms of VEGF-A signaling in BM-EPCs during hypoxia that may be important for migration to sites of vascular regeneration.

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Abstract 1743: Stem/Progenitor Markers Sca-1 versus C-kit and CD31 Determine Angiogenic Potential of Mouse Bone Marrow-derived Endothelial Progenitor Cells

Circulation ◽

10.1161/circ.118.suppl_18.s_381 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Toshikazu D Tanaka ◽

Masaaki Ii ◽

Haruki Sekiguchi ◽

Kentaro Jujo ◽

Sol Misener ◽

...

Keyword(s):

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Mononuclear Cells ◽

Tube Formation ◽

Mouse Bone Marrow ◽

Endothelial Progenitor ◽

Angiogenic Potential ◽

Ischemic Tissue

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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Establishment of Tube Formation Assay of Bone Marrow-Derived Endothelial Progenitor Cells

CNS Neuroscience & Therapeutics ◽

10.1111/cns.12102 ◽

2013 ◽

Vol 19 (7) ◽

pp. 533-535 ◽

Cited By ~ 9

Author(s):

Ji-Kuai Chen ◽

Ya-Ping Deng ◽

Guo-Jun Jiang ◽

Yun-Zi Liu ◽

Ting Zhao ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Tube Formation ◽

Endothelial Progenitor ◽

Tube Formation Assay

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PPARα Is Essential for Microparticle-Induced Differentiation of Mouse Bone Marrow-Derived Endothelial Progenitor Cells and Angiogenesis

PLoS ONE ◽

10.1371/journal.pone.0012392 ◽

2010 ◽

Vol 5 (8) ◽

pp. e12392 ◽

Cited By ~ 38

Author(s):

Tarek Benameur ◽

Simon Tual-Chalot ◽

Ramaroson Andriantsitohaina ◽

María Carmen Martínez

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Mouse Bone Marrow ◽

Endothelial Progenitor ◽

Induced Differentiation

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Mouse Bone Marrow-Derived Endothelial Progenitor Cells Do Not Restore Radiation-Induced Microvascular Damage

ISRN Cardiology ◽

10.1155/2014/506348 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 8

Author(s):

Ingar Seemann ◽

Johannes A. M. te Poele ◽

Saske Hoving ◽

Fiona A. Stewart

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Endothelial Damage ◽

Endothelial Cell Proliferation ◽

Mouse Bone Marrow ◽

Endothelial Progenitor ◽

Microvascular Damage ◽

Radiation Induced ◽

Microvasculature Damage

Background. Radiotherapy is commonly used to treat breast and thoracic cancers but it also causes delayed microvascular damage and increases the risk of cardiac mortality. Endothelial cell proliferation and revascularization are crucial to restore microvasculature damage and maintain function of the irradiated heart. We have therefore examined the potential of bone marrow-derived endothelial progenitor cells (BM-derived EPCs) for restoration of radiation-induced microvascular damage. Material & Methods. 16 Gy was delivered to the heart of adult C57BL/6 mice. Mice were injected with BM-derived EPCs, obtained from Eng+/+ or Eng+/− mice, 16 weeks and 28 weeks after irradiation. Morphological damage was evaluated at 40 weeks in transplanted mice, relative to radiation only and age-matched controls. Results. Cardiac irradiation decreased microvascular density and increased endothelial damage in surviving capillaries (decrease alkaline phosphatase expression and increased von Willebrand factor). Microvascular damage was not diminished by treatment with BM-derived EPCs. However, BM-derived EPCs from both Eng+/+ and Eng+/− mice diminished radiation-induced collagen deposition. Conclusion. Treatment with BM-derived EPCs did not restore radiation-induced microvascular damage but it did inhibit fibrosis. Endoglin deficiency did not impair this process.

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Delta Like 4 Expressing Bone Marrow-Derived Endothelial Progenitor Cells Regulate Tumour Angiogenesis

Blood ◽

10.1182/blood.v110.11.3728.3728 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3728-3728

Author(s):

Carla Real ◽

Francisco Caiado ◽

Catia Igreja ◽

Ana P. Elias ◽

Cristina Borges ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Therapeutic Potential ◽

Tumor Vasculature ◽

Endothelial Damage ◽

Endothelial Differentiation ◽

Endothelial Progenitor ◽

Vessel Stability

Abstract Bone marrow-derived endothelial progenitor cells (BM-EPCs) have been implicated in adult neoangiogenesis and consequently used as therapies for human pathologies with endothelial damage. The administration of these cells in human patients temporally improves endothelial function, although the engraftment of these cells in newly formed vessels is inefficient. Conversely, therapeutic stratagies to block EPC contribution during tumor angiogenesis have been proposed. In this work, we analysed the role of the Notch/Delta signalling pathway in EPC function during tumour neoangiogenesis, by regulating the expression of Notch ligand, delta-like 4 (Dll4) in these cells. Sublethally irradiated NOD-SCID mice received WT, Dll4+/− (Dll4 heterozygous mice) or Dll4 SiRNA-treated BM-EPCs and were subcutaneously inoculated with well established Human or murine tumor xenografts. Tumours growing in Dll4-depleted EPCs transplanted mice presented increased microvessel density when compared with WT EPCs transplanted mice or non-transplanted controls, regardless of VEGF expression. Although with increased vessel number, tumours of Dll4+/− EPC transplanted mice presented increased hypoxia and decreased tumour cell proliferation, suggesting an impairment in vessel function. In addition, these tumours present a diminished expression of PDGF, a vessel stabilizing factor, and increased expression of Ang2, known as a vessel destabilizing factor. We next verified whether the vessel destabilization observed in tumors after Dll4-depleted EPCs transplant might be due to a diferential endothelial differentiation or incorporation of EPCs in the tumour vasculature. In order to answer this question we quantified the incorporation of WT and Dll4-depleted EPCs in tumour vessels. Accordingly to our results, the presence of Dll4-depleted EPCs was reduced compared to WT EPCs, suggesting that Dll4-depleted EPCs might have reduced capacity to adhere to the renewing tumor vasculature, or to the underlying basement membrane. To test this, we used an in vitro endothelial differentiation assay, and observed a defect on the adhesion of of Dll4-depleted EPCs to extracellular matrix, which was correlated with a reduced expression of integrin subunits a3 and b1. These results suggest that the reduction of Dll4 on EPCs reduces integrin expression interfering with their ability to adhere, incorporate and stabilize the tumor vasculature during tumor neoangiogenesis. Therefore, EPCs have a major role in vessel stabilization in active neoangiogenic sites by the regulation of Dll4 expression. We propose that targeting the Notch/Dll4 pathway on EPCs, modulating vessel stability, may have therapeutic potential.

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Abstract P375: Gender-based Cardio-protective Functional Dimorphism Of Bone Marrow Endothelial Progenitor Cells And Their Exosomes: Estrogen-independent Epigenetic Mechanisms

Circulation Research ◽

10.1161/res.129.suppl_1.p375 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Charan T Gurrala ◽

Venkata Garikipati ◽

Zhongjian Cheng ◽

Vandana Mallaredy ◽

Maria Cimini ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Capillary Density ◽

Tube Formation ◽

Ischemic Myocardium ◽

Epigenetic Mechanism ◽

Epigenetic Mechanisms ◽

Endothelial Progenitor

Introduction: Estrogen or estrogen receptor-dependent mechanisms in enhancing the cardioprotective efficacy of bone marrow endothelial progenitor cells (BM-EPC) is well-established in preclinical studies. However, the efficacy of estrogen does not reflect in the data from randomized cardiovascular clinical trials, suggesting an estrogen-independent role of female BM-EPC in eliciting enhanced cardiac protection compared to males. Hypothesis: Epigenetic mechanisms may contribute to the sex-specific dimorphism of Sca-1 + /CD31 + BM-EPC in regulating cell-homing, pro-angiogenic and anti-inflammatory functions in the ischemic myocardium leading to enhanced reparative function of female progenitor cells. Methods & Results: Transplantation of GFP-BM-mononuclear cells from male and female GFP transgenic mice into the BM of lethally irradiated recipient male C57BL/6 mice resulted in the enhanced mobilization of female Sca-1 + CD31 + /GFP + BM-EPC into circulation post-MI. A higher number of female BM-EPC homed to the ischemic myocardium and significantly improved LV functions and capillary density post-MI compared to male BM-EPC. Female BM-EPC showed increased expression of bFGF, VEGFR2, SDF-1α, and IL-10 genes, thereby efficiently promoted endothelial tube formation in vitro compared to male BM-EPC. Transplantation of female BM-EPC and their exosomes into post-MI male mice improved LV cardiac function, reduced scar size, and improved capillary density compared to male BM-EPC and exosomes. Male BM-EPC showed an increased expression of G9a/Ehmt2, an H3K9me3 methyltransferase, and Dnmt3a DNA methyltransferase compared to female BM-EPC. In contrast, Kdm6b/JMJD3, H3K27me3 demethylase was highly expressed in female BM-EPC compared to males. Treatment of BM-EPC of both sexes with 17-β-estradiol did not alter the expression of Kdm6b/JMJD3. Male BM-EPC highly expressed repressive gene marks, H3K9me3, and H3K27me3 compared to females. Compared to the male, BM-EPC from female and ovariectomized (OXV) female mice showed equally high expression of angiogenic genes ANGPT-1, MDK, PLAU, Tie-2, and VEGFR2 and lower levels of inflammatory cytokines, TNFα, IFNγ, IL-1β, and CCL3. Conditioned medium from female and OVX BM-EPC equally promoted enhanced migration and tube formation of HUVEC in vitro, compared to male BM-EPC. Conclusions: An estrogen-independent epigenetic mechanism likely governs the enhanced cardiac reparative properties of female BM-EPC.

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