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1.  Actin Stabilization by Jasplakinolide Affects the Function of Bone Marrow-Derived Late Endothelial Progenitor Cells 
PLoS ONE  2012;7(11):e50899.
Background
Bone marrow-derived endothelial progenitor cells (EPCs), especially late EPCs, play a critical role in endothelial maintenance and repair, and postnatal vasculogenesis. Although the actin cytoskeleton has been considered as a modulator that controls the function and modulation of stem cells, its role in the function of EPCs, and in particular late EPCs, remains poorly understood.
Methodology/Principal Finding
Bone marrow-derived late EPCs were treated with jasplakinolide, a compound that stabilizes actin filaments. Cell apoptosis, proliferation, adhesion, migration, tube formation, nitric oxide (NO) production and endothelial NO synthase (eNOS) phosphorylation were subsequently assayed in vitro. Moreover, EPCs were locally infused into freshly balloon-injured carotid arteries, and the reendothelialization capacity was evaluated after 14 days. Jasplakinolide affected the actin distribution of late EPCs in a concentration and time dependent manner, and a moderate concentration of (100 nmol/l) jasplakinolide directly stabilized the actin filament of late EPCs. Actin stabilization by jasplakinolide enhanced the late EPC apoptosis induced by VEGF deprivation, and significantly impaired late EPC proliferation, adhesion, migration and tube formation. Furthermore, jasplakinolide attenuated the reendothelialization capacity of transplanted EPCs in the injured arterial segment in vivo. However, eNOS phosphorylation and NO production were increased in late EPCs treated with jasplakinolide. NO donor sodium nitroprusside (SNP) rescued the functional activities of jasplakinolide-stressed late EPCs while the endothelial NO synthase inhibitor L-NAME led to a further dysfunction induced by jasplakinolide in late EPCs.
Conclusions/Significance
A moderate concentration of jasplakinolide results in an accumulation of actin filaments, enhancing the apoptosis induced by cytokine deprivation, and impairing the proliferation and function of late EPCs both in vitro and in vivo. NO donor reverses these impairments, suggesting the role of NO-related mechanisms in jasplakinolide-induced EPC downregulation. Actin cytoskeleton may thus play a pivotal role in regulating late EPC function.
doi:10.1371/journal.pone.0050899
PMCID: PMC3511387  PMID: 23226422
2.  Transplantation of cryopreserved human umbilical cord blood-derived endothelial progenitor cells induces recovery of carotid artery injury in nude rats 
Introduction
Transplantation of endothelial progenitor cells (EPCs) restores endothelial function in patients with endothelial dysfunction and initial denudation. The goal of the present study was to determine the effect of cryopreserved human umbilical cord blood (UCB)-derived EPC infusion on the repair of carotid artery injury in nude rats.
Methods
Mononuclear cells (MNCs) from human cryopreserved UCB and peripheral blood (PB) of patients with cardiovascular diseases and healthy volunteers were cultured in a conditioned medium. The in vitro migration, proliferation, adhesion, and survival capacities, as well as paracrine cytokine release of EPCs were investigated. EPC homing, induced reendothelialization, and the effect on neointima formation were also assessed in vivo.
Results
Patient-derived PB EPCs (PPB-EPCs) displayed decreased migration, proliferation, adhesion, and survival capabilities as compared to PB-EPCs from healthy volunteers (HPB-EPCs) and cryopreserved UCB-EPCs. However, there was no difference in the release of vascular endothelial growth factor (VEGF) and stromal cell derived factor 1 (SDF-1) between the three groups. Two weeks after transplantation, more labeled UCB-EPCs and HPB-EPCs than PPB-EPCs were found by cell tracking in the injury zone. Administration of PPB-EPCs, HPB-EPCs, and UCB-EPCs enhanced reendothelialization and inhibited neointima formation compared to the saline control. However, UCB-EPC and HPB-EPC infusion showed a greater improvement than PPB-EPCs.
Conclusions
Cryopreserved UCB-MNCs derived EPCs and HPB-EPCs show better responses to cytokines and vascular injury than PPB-EPCs. Thus, cryopreservation and delivery of cryopreserved autogenous UCB-EPCs or HPB-EPCs may be a promising vasculoprotective approach for patients with multiple cardiovascular risk factors.
Electronic supplementary material
The online version of this article (doi:10.1186/s13287-015-0022-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13287-015-0022-4
PMCID: PMC4453210  PMID: 25956351
3.  Identification of mouse colony-forming endothelial progenitor cells for postnatal neovascularization: a novel insight highlighted by new mouse colony-forming assay 
Introduction
Endothelial progenitor cells (EPCs) play a critical role in restoration of ischemic diseases. However, the actual status of EPC development and the mechanisms of EPC dysfunctions in patients with various ischemic diseases remain unknown.
Methods
To investigate the detailed function of EPCs in experimental murine models, we have established an EPC colony forming assay (EPC-CFA) in murine EPCs. The abilities of murine EPCs in differentiation, adhesive capacity, proliferative potency, and transplantation in vitro and in vivo were then examined.
Results
Peripheral blood mononuclear cells (PB-MNCs), bone marrow mononuclear cells (BM-MNCs) or bone marrow c-Kit+/Sca-1+ lineage negative (BM-KSL) cells differentiated into two types of EPC colony forming units (EPC-CFUs), large sized EPC (large-EPC)-CFUs and small sized EPC (small-EPC)-CFUs. Gene expression analysis demonstrated that both EPC-CFU-derived cells expressed eNOS, Flk-1 and VE-cadherin, markers of endothelial cells (ECs), although the small-EPCs derived from small-EPC-CFU were higher in number and showed more immature features (higher population of KSL cells). Functionally, the large-EPCs derived from large-EPC-CFU had higher adhesive capacity but lower proliferative potency than small-EPCs, showing improved tubular forming capacity and incorporation potency into primary EC-derived tube formation. Importantly, hindlimb ischemia increased the frequencies of large-EPC-CFUs differentiated from PB-MNCs and bone marrow. Actually, transplantation of large-EPCs into ischemic hindlimb enhanced neovascularization in hindlimb ischemia model, although small-EPCs or murine ECs did not, suggesting that large-EPC-CFUs might play an important role in restoration of ischemic diseases.
Conclusions
We demonstrated, using a murine ischemia model, that the EPC-CFA could be a useful way to investigate the differentiation levels of murine EPCs, further providing a crucial clue that large-EPC-CFU status may be more functional or effective EPCs to promote neovascularization.
doi:10.1186/scrt168
PMCID: PMC3706928  PMID: 23448126
4.  Rosiglitazone via upregulation of Akt/eNOS pathways attenuates dysfunction of endothelial progenitor cells, induced by advanced glycation end products 
British Journal of Pharmacology  2009;158(8):1865-1873.
Background and purpose:
Advanced glycation end products (AGEs) and endothelial progenitor cells (EPCs) play key roles in pathogenesis of diabetes-related vascular complications. AGEs can induce dysfunction in EPCs. The peroxisome proliferator-activated receptor-gamma (PPARγ) agonists are widely used in the treatment of type 2 diabetes, and it remains unknown if they could attenuate EPC dysfunction induced by AGEs.
Experimental approach:
EPCs isolated from healthy adults were cultured with various concentrations of AGEs (0, 50, 100 and 200 mg·L−1) with or without rosiglitazone (10 nM), antibody for the receptors for AGE-human serum albumin (anti-receptor for advanced glycation end products (RAGE); 50 µg·mL−1), phosphatidylinositol-3-kinase (PI3K) inhibitor (LY294002, 5 µM), nitric oxide (NO) synthase inhibitor (L-NG-nitro-arginine methyl ester (L-NAME), 100 µM) or sodium nitroprusside (SNP, 25 µM). Proliferation, apoptosis, cell adhesion, migration and NO production in EPCs were assessed, and expressions of endothelial NO synthase (eNOS) and Akt were determined.
Key results:
Number, proliferation/migration capacities, eNOS and Akt phosphorylation as well as NO synthesized by EPCs were increased by rosiglitazone and reduced by AGEs. AGEs promoted while rosiglitazone reduced EPC apoptosis. The AGE-induced effects were significantly ameliorated by pre-incubation with rosiglitazone, RAGE antibody and SNP. The beneficial effects of rosiglitazone could be blocked by pretreatment with L-NAME and LY294002.
Conclusions and implications:
The PPARγ agonist rosiglitazone increased EPC function and attenuated EPC dysfunction induced by AGEs via upregulating the Akt-eNOS signal pathways of EPCs.
doi:10.1111/j.1476-5381.2009.00450.x
PMCID: PMC2807648  PMID: 19917066
endothelial progenitor cells; advanced glycation end products; receptor for advanced glycation end products; apoptosis; migration; peroxisome proliferator-activated receptor-γ agonists
5.  Validation study to compare effects of processing protocols on measured Nε-(carboxymethyl)lysine and Nε-(carboxyethyl)lysine in blood 
Epidemiological studies show that elevated plasma levels of advanced glycation end products (AGEs) are associated with diabetes, kidney disease, and heart disease. Thus AGEs have been used as disease progression markers. However, the effects of variations in biological sample processing procedures on the level of AGEs in plasma/serum samples have not been investigated. The objective of this investigation was to assess the effect of variations in blood sample collection on measured Nε-(carboxymethyl)lysine (CML), the best characterised AGE, and its homolog, Nε-(carboxyethyl)lysine (CEL). The investigation examined the effect on CML and CEL of different blood collection tubes, inclusion of a stabilising cocktail, effect of freeze thaw cycles, different storage times and temperatures, and effects of delaying centrifugation on a pooled sample from healthy volunteers. CML and CEL were measured in extracted samples by ultra-performance liquid chromatography-tandem mass spectrometry. Median CML and CEL ranged from 0.132 to 0.140 mM/M lys and from 0.053 to 0.060 mM/M lys, respectively. No significant difference was shown CML or CEL in plasma/serum samples. Therefore samples collected as part of epidemiological studies that do not undergo specific sample treatment at collection are suitable for measuring CML and CEL.
doi:10.3164/jcbn.13-5
PMCID: PMC3818270  PMID: 24249965
advanced glycation end-products; Nε-(carboxymethyl)lysine; Nε-(carboxyethyl)lysine; epidemiology; blood sampling
6.  Downregulation of MicroRNA-130a Contributes to Endothelial Progenitor Cell Dysfunction in Diabetic Patients via Its Target Runx3 
PLoS ONE  2013;8(7):e68611.
Dysfunction of endothelial progenitor cells (EPCs) contributes to diabetic vascular disease. MicroRNAs (miRs) have emerged as key regulators of diverse cellular processes including angiogenesis. We recently reported that miR-126, miR-130a, miR-21, miR-27a, and miR-27b were downregulated in EPCs from type II diabetes mellitus (DM) patients, and downregulation of miR-126 impairs EPC function. The present study further explored whether dysregulated miR-130a were also related to EPC dysfunction. EPCs were cultured from peripheral blood mononuclear cells of diabetic patients and healthy controls. Assays on EPC function (proliferation, migration, differentiation, apoptosis, and colony and tubule formation) were performed. Bioinformatics analyses were used to identify the potential targets of miR-130a in EPCs. Gene expression of miR-103a and Runx3 was measured by real-time PCR, and protein expression of Runx3, extracellular signal-regulated kinase (ERK), vascular endothelial growth factor (VEGF) and Akt was measured by Western blotting. Runx3 promoter activity was measured by luciferase reporter assay. A miR-130a inhibitor or mimic and lentiviral vectors expressing miR-130a, or Runx3, or a short hairpin RNA targeting Runx3 were transfected into EPCs to manipulate miR-130a and Runx3 levels. MiR-130a was decreased in EPCs from DM patients. Anti-miR-130a inhibited whereas miR-130a overexpression promoted EPC function. miR-130a negatively regulated Runx3 (mRNA, protein and promoter activity) in EPCs. Knockdown of Runx3 expression enhanced EPC function. MiR-130a also upregulated protein expression of ERK/VEGF and Akt in EPCs. In conclusion, miR-130a plays an important role in maintaining normal EPC function, and decreased miR-130a in EPCs from DM contributes to impaired EPC function, likely via its target Runx3 and through ERK/VEGF and Akt pathways.
doi:10.1371/journal.pone.0068611
PMCID: PMC3709913  PMID: 23874686
7.  Transfusion of CXCR4-Primed Endothelial Progenitor Cells Reduces Cerebral Ischemic Damage and Promotes Repair in db/db Diabetic Mice 
PLoS ONE  2012;7(11):e50105.
This study investigated the role of stromal cell-derived factor-1α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) axis in brain and endothelial progenitor cells (EPCs), and explored the efficacy of CXCR4 primed EPCs in treating ischemic stroke in diabetes. The db/db diabetic and db/+ mice were used in this study. Levels of plasma SDF-1α and circulating CD34+CXCR4+ cells were measured. Brain SDF-1α and CXCR4 expression were quantified at basal and after middle cerebral artery occlusion (MCAO). In in vitro study, EPCs were transfected with adenovirus carrying null (Ad-null) or CXCR4 (Ad-CXCR4) followed with high glucose (HG) treatment for 4 days. For pathway block experiments, cells were pre-incubated with PI3K inhibitor or nitric oxide synthase (NOS) inhibitor for two hours. The CXCR4 expression, function and apoptosis of EPCs were determined. The p-Akt/Akt and p-eNOS/eNOS expression in EPCs were also measured. In in vivo study, EPCs transfected with Ad-null or Ad-CXCR4 were infused into mice via tail vein. On day 2 and 7, the cerebral blood flow, neurologic deficit score, infarct volume, cerebral microvascular density, angiogenesis and neurogenesis were determined. We found: 1) The levels of plasma SDF-1α and circulating CD34+CXCR4+ cells were decreased in db/db mice; 2) The basal level of SDF-1α and MCAO-induced up-regulation of SDF-1α/CXCR4 axis were reduced in the brain of db/db mice; 3) Ad-CXCR4 transfection increased CXCR4 expression in EPCs and enhanced EPC colonic forming capacity; 4) Ad-CXCR4 transfection prevented EPCs from HG-induced dysfunction (migration and tube formation) and apoptosis via activation of PI3K/Akt/eNOS signal pathway; 4) Ad-CXCR4 transfection enhanced the efficacy of EPC infusion in attenuating infarct volume and promoting angiogenesis and neurogenesis. Our data suggest that Ad-CXCR4 primed EPCs have better therapeutic effects for ischemia stroke in diabetes than unmodified EPCs do.
doi:10.1371/journal.pone.0050105
PMCID: PMC3503762  PMID: 23185548
8.  IL-10 deficiency impairs bone marrow-derived endothelial progenitor cell (EPC) survival and function in ischemic myocardium 
Circulation research  2011;109(11):1280-1289.
Rationale
Endothelial progenitor cell (EPC) survival and function in the injured myocardium is adversely influenced by hostile microenvironment like ischemia, hypoxia and inflammatory response, thereby compromising full benefits of EPC-mediated myocardial repair.
Objective
We hypothesized that interleukin-10 (IL-10) modulates EPC biology leading to enhanced survival and function following transplantation in the ischemic myocardium.
Methods and Results
Myocardial infarction (MI)-induced mobilization of bone marrow EPC (Sca-1+Flk1+ cells) into the circulation was significantly impaired in IL-10 KO-mice. Bone marrow transplantation (BMT) to replace IL-10 KO-marrow with WT-marrow attenuated these effects. Impaired mobilization was associated with lower SDF-1 expression levels in the myocardium of KO-mice. Interestingly, SDF-1 administration reversed mobilization defect in KO-mice. In vitro, hypoxia-mediated increases in CXCR4 expression and cell survival were lower in IL-10-deficient-EPCs. Furthermore, SDF-1-induced migration of WT-EPCs was inhibited by AMD3100, an inhibitor of CXCR4. To further study the effect of IL-10 on in vivo EPC survival and engraftment into vascular structures, GFP-labeled EPC were injected intramyocardially after induction of MI, and the mice were treated with either saline or recombinant IL-10. IL-10-treated group showed increased retention of transplanted EPCs in the myocardium and was associated with significantly reduced EPC apoptosis post-MI. Interestingly, increased EPC retention and their association with the vascular structures was observed in IL-10 treated mice. Increased EPC survival and angiogenesis in the myocardium of IL-10-treated mice corroborated with improved LV function, reduced infarct size and fibrosis in the myocardium. In vitro, IL-10-induced increase in VEGF expression in WT-EPC was abrogated by STAT3 inhibitor suggesting IL-10 signals via STAT3 activation.
Conclusions
Taken together, our studies demonstrate that MI-induced EPC mobilization was impaired in IL-10 KO-mice and that IL-10 increases EPC survival and function possibly via activation of STAT3/VEGF signaling cascades, leading to attenuation of MI-induced LV dysfunction and remodeling.
doi:10.1161/CIRCRESAHA.111.248369
PMCID: PMC3235675  PMID: 21959218
Endothelial progenitor cells; survival; myocardial infarction; IL-10; bone marrow transplantation; inflammation; angiogenesis; left ventricular remodeling
9.  Zoledronate Attenuates Angiogenic Effects of Angiotensin II-Stimulated Endothelial Progenitor Cells via RhoA and MAPK Signaling 
PLoS ONE  2012;7(10):e46511.
Background
New vessel formation plays a pivotal role in the pathogenesis of neovascular-related diseases. Endothelial progenitor cells (EPCs) were found to contribute to neovascular-related diseases and interference with EPC neovascularization may be a novel target for these diseases. Zoledronate (Zol) was reported to exhibit anti-angiogenic effect. Basing on these evidences, we proposed that Zol may affect EPC function to exert novel anti-angiogenic effect. In this study, we therefore investigated the effects of Zol on multiple aspects of EPC function and explored the underlying mechanisms involved.
Methodology/Principal Findings
EPCs were cultured from bone marrow derived mononuclear cells. The potential effects of Zol on Angiotensin II (Ang II)-stimulated EPC proliferation, migration, adhesion, in vitro tube formation were investigated. The results showed that Ang II (1 µM) enhanced EPC migration, adhesion, in vitro tube formation but had no effect on cell proliferation. Zol (75 and 100 µM) inhibited proliferation of EPCs and 50 µM geranylgeranyol (GGOH) could reverse the decrease of EPC proliferation. We found for the first time that Zol (50–100 µM) dose dependently attenuated migration, adhesion, and in vitro tube formation of EPCs stimulated by Ang II. GGOH could reverse the attenuation of EPC function induced by Zol. However, Zol did not induce EPC apoptosis. In addition, the underlying mechanisms were determined. The results revealed that Zol markedly down-regulated active RhoA stimulated by Ang II and inhibited the phosphorylation of Erk1/2 and JNK. Moreover, RhoA silencing resulted in a notable inhibition of EPC in vitro tube formation, suggesting that RhoA suppression played a pivotal role in Zol antiangiogenic effect.
Conclusions/Significance
These findings suggested that Zol attenuated the promotion of EPC function stimulated by Ang II and exhibited novel antiangiogenic effect via RhoA and MAPK signaling. Thus, Zol may be served as a novel therapeutic agent for neovascular-related diseases treatment.
doi:10.1371/journal.pone.0046511
PMCID: PMC3469623  PMID: 23071580
10.  Advanced glycation end products impair the migration, adhesion and secretion potentials of late endothelial progenitor cells 
Background
Endothelial progenitor cells (EPCs), especially late EPCs, play a critical role in endothelial maintenance and repair, and postnatal vasculogenesis. Advanced glycation end products (AGEs) have been shown to impair EPC functions, such as proliferation, migration and adhesion. However, their role in the regulation of the production of vasoactive substances in late EPCs is less well defined.
Methods
Passages of 3~5 EPCs, namely late EPCs, were cultured with different concentrations (0~500 μg/ml) of AGEs, and the apoptosis, adhesion and migration were subsequently determined. The release of vasoactive substances, such as stromal cell-derived factor-1 (SDF-1), nitric oxide (NO), prostaglandin I2 (PGI2), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), and in addition the activity of superoxide dismutase (SOD), were evaluated by ELISA. At the same time, the gene and protein expressions of CXCR4 were assayed by real-time RT-PCR and western-blot.
Results
AGEs promoted late EPC apoptosis. Moreover, AGEs impaired late EPC migration and adhesion in a concentration-dependent manner. Accordingly, the production of SDF-1 was decreased by AGEs. Although the CXCR4 expressions of late EPCs were up-regulated for AGE concentrations of 50, 100 or 200 μg/ml, a marked decrease was observed for the higher concentration of 500 μg/ml. Furthermore, co-culturing with AGEs decreased the levels of NO, t-PA, PGI2, and the activity of SOD but up-regulated the production of PAI-1.
Conclusion
Our data provide evidence that AGEs play an important role in impairing late EPC functions, which could contribute to the development of vascular diseases in diabetes.
doi:10.1186/1475-2840-11-46
PMCID: PMC3403843  PMID: 22545734
Endothelial progenitor cells; AGEs; Diabetes; Vasoactive substances
11.  Cleaved high molecular weight kininogen inhibits tube formation of endothelial progenitor cells via suppression of matrix metalloproteinase 2 
Background and objective
Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, thus promoting wide interest in their therapeutic potential in vascular injury and prevention of their dysfunction in cardiovascular diseases. Cleaved high molecular weight kininogen (HKa), an activation product of the plasma kallikrein-kinin system (KKS), inhibits the functions of differentiated endothelial cells including in vitro and in vivo angiogenesis. In this study, our results provided the first evidence that HKa is able to target EPCs and inhibits their tube forming capacity.
Methods and results
We determined the effect of HKa on EPCs using a three-dimensional vasculogenesis assay. Upon stimulation with vascular endothelial growth factor (VEGF) alone, EPCs formed vacuoles and tubes, and differentiated into capillary-like networks. As detected by gelatinolytic activity assay, VEGF stimulated secretion and activation of matrix metallopeptidase 2 (MMP-2), but not MMP-9, in the conditioned medium of 3D culture of EPCs. Specific inhibition or gene ablation of MMP-2, but not MMP-9, blocked the vacuole and tube formation by EPCs. Thus, MMP-2 is selectively required for EPC vasculogenesis. In a concentration-dependent manner, HKa significantly inhibited tube formation by EPCs and the conversion of pro-MMP-2 to MMP-2. Moreover, HKa completely blocked the association between pro-MMP- 2 and αvβ3 integrin, and its inhibition of MMP-2 activation was dependent on the presence of αvβ3 integrin. In a purified system, HKa did not directly inhibit MMP-2 activity.
Conclusions
HKa inhibits tube forming capacity of EPCs by suppression of MMP-2 activation, which may constitute a novel link between activation of the KKS and EPC dysfunction.
doi:10.1111/j.1538-7836.2009.03662.x
PMCID: PMC3142619  PMID: 19874467
endothelial progenitor cells; kininogen; matrix metalloproteinase; vasculogenesis
12.  INFLUENCE OF ELEVATED LEVELS OF C-REACTIVE PROTEIN ON CIRCULATING ENDOTHELIAL PROGENITOR CELL FUNCTION 
In vitro, C-reactive protein (CRP) impairs endothelial progenitor cell (EPC) function; however, the influence of CRP on EPCs in vivo is unclear. We determined whether EPC function is impaired in adults with elevated plasma CRP concentrations, independent of other risk factors. EPCs were harvested from 75 adults (43 males, 32 females): 25 with low CRP (< 1.0 mg/L); 25 with moderate CRP (1.0–3.0 mg/L); and 25 with high CRP (> 3.0 mg/L). The capacity of EPCs to form colonies (colony assay), migrate (Boyden chamber), release angiogenic growth factor (ELISA) and resist apoptosis (active caspase-3) was determined. There were no significant differences between the CRP groups in EPC colony formation (CFU), migration (AU) or the ability to release vascular endothelial growth factor (VEGF; pg/mL): low (13±3 CFU; 1255±100 AU; 126±24 pg/mL); moderate (11±3 CFU; 1137±85 AU; 97±14 pg/mL); and high (13±4 CFU; 1071±80 AU; 119±22 pg/mL) CRP. Staurosporine-stimulated activation of caspase-3 was also similar between the low (2.3±0.2 ng/mL), moderate (2.1±0.3 ng/mL), and high (2.2±0.2 ng/mL) CRP groups. These results indicate that elevations in plasma CRP are not associated with impaired EPC function. EPC dysfunction may not play a role in CRP-related cardiovascular risk.
doi:10.1111/cts.12137
PMCID: PMC4113468  PMID: 24422709
13.  Novel Cell-Free Strategy for Therapeutic Angiogenesis: In Vitro Generated Conditioned Medium Can Replace Progenitor Cell Transplantation 
PLoS ONE  2009;4(5):e5643.
Background
Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy.
Methodology/Principal Findings
EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2±2.9% and 83.7±3.0% vs. 53.5±2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62±0.03 and 1.68±0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6±0.3 and 8.1±0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7±44.1 vs. 340.0±29.1 CD34+/CD45− cells/1×105 mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9±0.7 vs. 2.6±0.4 CD34+ cells/HPF, P<0.001) 3 days after the last injection.
Conclusions/Significance
Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.
doi:10.1371/journal.pone.0005643
PMCID: PMC2682571  PMID: 19479066
14.  Dextran induces differentiation of circulating endothelial progenitor cells 
Physiological Reports  2014;2(3):e00261.
Abstract
Endothelial progenitor cells (EPCs) have been demonstrated to be effective for the treatment of cardiovascular diseases. However, the differentiation process from circulation to adhesion has not been clarified because circulating EPCs rarely attached to dishes in EPC cultures previously. Here we investigated whether immature circulating EPCs differentiate into mature adhesive EPCs in response to dextran. When floating‐circulating EPCs derived from ex vivo expanded human cord blood were cultured with 5% and 10% dextran, they attached to fibronectin‐coated dishes and grew exponentially. The bioactivities of adhesion, proliferation, migration, tube formation, and differentiated type of EPC colony formation increased in EPCs exposed to dextran. The surface protein expression rate of the endothelial markers vascular endothelial growth factor (VEGF)‐R1/2, VE‐cadherin, Tie2, ICAM1, VCAM1, and integrin αv/β3 increased in EPCs exposed to dextran. The mRNA levels of VEGF‐R1/2, VE‐cadherin, Tie2, endothelial nitric oxide synthase, MMP9, and VEGF increased in EPCs treated with dextran. Those of endothelium‐related transcription factors ID1/2, FOXM1, HEY1, SMAD1, FOSL1, NFkB1, NRF2, HIF1A, EPAS1 increased in dextran‐treated EPCs; however, those of hematopoietic‐ and antiangiogenic‐related transcription factors TAL1, RUNX1, c‐MYB, GATA1/2, ERG, FOXH1, HHEX, SMAD2/3 decreased in dextran‐exposed EPCs. Inhibitor analysis showed that PI3K/Akt, ERK1/2, JNK, and p38 signal transduction pathways are involved in the differentiation in response to dextran. In conclusion, dextran induces differentiation of circulating EPCs in terms of adhesion, migration, proliferation, and vasculogenesis. The differentiation mechanism in response to dextran is regulated by multiple signal transductions including PI3K/Akt, ERK1/2, JNK, and p38. These findings indicate that dextran is an effective treatment for EPCs in regenerative medicines.
Circulating endothelial progenitor cells (EPCs) rarely attach to dishes. Here, we showed immature circulating EPCs differentiate into mature adhesive EPCs in response to dextran.
doi:10.1002/phy2.261
PMCID: PMC4002241  PMID: 24760515
Culture; endothelial progenitor cell; signal transduction; transcription
15.  Impairment of endothelial progenitor cell function and vascularization capacity by aldosterone in mice and humans 
European Heart Journal  2010;32(10):1275-1286.
Aims
Hyperaldosteronism is associated with vascular injury and increased cardiovascular events. Bone marrow-derived endothelial progenitor cells (EPCs) play an important role in endothelial repair and vascular homeostasis. We hypothesized that hyperaldosteronism impairs EPC function and vascularization capacity in mice and humans.
Methods and results
We characterized the effects of aldosterone and mineralocorticoid receptor (MR) blockade on EPC number and function as well as vascularization capacity and endothelial function. Treatment of human EPC with aldosterone induced translocation of the MR and impaired multiple cellular functions of EPC, such as differentiation, migration, and proliferation in vitro. Impaired EPC function was rescued by pharmacological blockade or genetic ablation of the MR. Aldosterone protein kinase A (PKA) dependently increased reactive oxygen species formation in EPC. Aldosterone infusion in mice impaired EPC function, EPC homing to vascular structures and vascularization capacity in a MR-dependent but blood pressure-independent manner. Endothelial progenitor cells from patients with primary hyperaldosteronism compared with controls of similar age displayed reduced migratory potential. Impaired EPC function was associated with endothelial dysfunction. MR blockade in patients with hyperaldosteronism improved EPC function and arterial stiffness.
Conclusion
Endothelial progenitor cells express a MR that mediates functional impairment by PKA-dependent increase of reactive oxygen species. Normalization of EPC function may represent a novel mechanism contributing to the beneficial effects of MR blockade in cardiovascular disease prevention and treatment.
doi:10.1093/eurheartj/ehq254
PMCID: PMC3094546  PMID: 20926363
Aldosterone; Primary hyperaldosteronism; Endothelial progenitor cells; Endothelial function; Reactive oxygen species
16.  Decreased Circulating Endothelial Progenitor Cell Levels and Function in Patients with Nonalcoholic Fatty Liver Disease 
PLoS ONE  2012;7(2):e31799.
Objectives
Nonalcoholic fatty liver disease (NAFLD) is associated with advanced atherosclerosis and a higher risk of cardiovascular disease. Increasing evidence suggests that injured endothelial monolayer is regenerated by circulating bone marrow derived-endothelial progenitor cells (EPCs), and levels of circulating EPCs reflect vascular repair capacity. However, the relation between NAFLD and EPC remains unclear. Here, we tested the hypothesis that patients with nonalcoholic fatty liver disease (NAFLD) might have decreased endothelial progenitor cell (EPC) levels and attenuated EPC function.
Methods and Results
A total of 312 consecutive patients undergoing elective coronary angiography because of suspected coronary artery disease were screened and received examinations of abdominal ultrasonography between July 2009 and November 2010. Finally, 34 patients with an ultrasonographic diagnosis of NAFLD, and 68 age- and sex-matched controls without NAFLD were enrolled. Flow cytometry with quantification of EPC markers (defined as CD34+, CD34+KDR+, and CD34+KDR+CD133+) in peripheral blood samples was used to assess circulating EPC numbers. The adhesive function, and migration, and tube formation capacities of EPCs were also determined in NAFLD patients and controls. Patients with NAFLD had a significantly higher incidence of metabolic syndrome, previous myocardial infarction, hyperuricemia, and higher waist circumference, body mass index, fasting glucose and triglyceride levels. In addition, patients with NAFLD had significantly decreased circulating EPC levels (all P<0.05), attenuated EPC functions, and enhanced systemic inflammation compared to controls. Multivariate logistic regression analysis showed that circulating EPC level (CD34+KDR+ [cells/105 events]) was an independent reverse predictor of NAFLD (Odds ratio: 0.78; 95% confidence interval: 0.69–0.89, P<0.001).
Conclusions
NAFLD patients have decreased circulating EPC numbers and functions than those without NAFLD, which may be one of the mechanisms to explain atherosclerotic disease progression and enhanced cardiovascular risk in patients with NAFLD.
doi:10.1371/journal.pone.0031799
PMCID: PMC3280999  PMID: 22359630
17.  Lovastatin restores the function of endothelial progenitor cells damaged by oxLDL 
Acta Pharmacologica Sinica  2009;30(5):545-552.
Aim:
The aim of the study was to investigate whether lovastatin restores the survival and function of endothelial progenitor cells (EPCs) damaged by oxLDL.
Methods:
EPCs were preincubated with different concentrations of lovastatin (2, 10, and 50 μmol/L) with or without the Akt inhibitor triciribine for 24 h and were then exposed to 50 μg/mL oxLDL for 48 h. The survival of EPCs, as well as the cellular migration, adhesion, and tube formation of these cells, was examined. To explore the mechanisms of lovastatin's effects on EPCs, the levels of phosphorylated Akt and eNOS and of total eNOS protein and mRNA were assayed.
Results:
Incubation of EPCs with oxLDL resulted in significant apoptosis and impaired cellular migration, adhesion and tube structure formation. The detrimental effects of oxLDL on EPC survival and function were attenuated by pretreatment of EPCs with lovastatin. However, when EPCs were pretreated with lovastatin and triciribine at the same time, the beneficial effects of lovastatin were abolished by triciribine. Furthermore, oxLDL caused a significant downregulation of eNOS mRNA and protein expression, as well as a suppression of Akt and eNOS phosphorylation. However, the effects of oxLDL on Akt/eNOS activity and eNOS expression were reversed by lovastatin.
Conclusion:
Lovastatin reverses the survival and function of EPCs by regulating the Akt/eNOS signaling pathway and the gene transcription of eNOS.
doi:10.1038/aps.2009.41
PMCID: PMC4002826  PMID: 19417733
oxidized low density lipoprotein; endothelial progenitor cells; endothelial nitric oxide synthase
18.  PREHYPERTENSION AND ENDOTHELIAL PROGENITOR CELL FUNCTION 
Journal of human hypertension  2010;25(1):57-62.
Prehypertension is associated with significant damage to the coronary vasculature and increased rates of adverse cardiovascular events. Circulating endothelial progenitor cells (EPCs) are critical to vascular repair and the formation of new blood vessels. We tested the hypothesis that prehypertension is associated with EPC dysfunction. Peripheral blood samples were collected from 83 middle-aged and older adults (51 M/32 F): 40 normotensive (age 53±2 yr; BP 111/74±1/1 mmHg) and 43 prehypertensive (54±2; 128/77±1/1 mmHg). EPCs were isolated from peripheral blood and EPC colony-forming capacity (colony-forming unit assay), migratory activity (Boyden chamber) and apoptotic susceptibility (active caspase-3 concentrations) were determined. There were no significant differences in either the number of EPC CFUs (10±2 vs. 9±1), EPC migration (1165±82 vs. 1120±84 fluorescent units), or active intracellular caspase-3 concentrations (2.7±0.3 vs. 2.3±0.2 ng/mL) between the normotensive and prehypertensive groups. When groups were stratified into low prehypertension (n=27; systolic BP: 120–129 mmHg) and high prehypertension (n=16; 130–139 mmHg), it was found that EPCs from the high prehypertensive group produced fewer (~65%, P<0.05) CFUs compared with the low prehypertensive (4±1 vs. 12±2) and normotensive adults. In conclusion, EPC colonyforming capacity is impaired only in prehypertensive adults with systolic BP greater than 130 mmHg. Prehypertension is not associated with migratory dysfunction or enhanced apoptosis of EPCs.
doi:10.1038/jhh.2010.31
PMCID: PMC2895004  PMID: 20336149
endothelial progenitor cells; prehypertension
19.  Diagnostic potential of plasma carboxymethyllysine and carboxyethyllysine in multiple sclerosis 
Background
This study compared the level of advanced glycation end products (AGEs), N-(Carboxymethyl)lysine (CML) and N-(Carboxyethyl)lysine (CEL), in patients with multiple sclerosis (MS) and healthy controls (HCs), correlating these markers with clinical indicators of MS disease severity.
Methods
CML and CEL plasma levels were analyzed in 99 MS patients and 43 HCs by tandem mass spectrometry (LC/MS/MS). Patients were stratified based on drug modifying therapies (DMTs) including interferon beta, glatiramer acetate and natalizumab.
Results
The level of plasma CEL, but not CML, was significantly higher in DMT-naïve MS patients when compared to HCs (P < 0.001). Among MS patients, 91% had higher than mean plasma CEL observed in HCs. DMTs reduced CML and CEL plasma levels by approximately 13% and 40% respectively. CML and CEL plasma levels correlated with the rate of MS clinical relapse.
Conclusion
Our results suggest that AGEs in general and CEL in particular could be useful biomarkers in MS clinical practice. Longitudinal studies are warranted to determine any causal relationship between changes in plasma level of AGEs and MS disease pathology. These studies will pave the way for use of AGE inhibitors and AGE-breaking agents as new therapeutic modalities in MS.
doi:10.1186/1742-2094-7-72
PMCID: PMC2984414  PMID: 21034482
20.  A role for plasma kallikrein-kinin system activation in the synovial recruitment of endothelial progenitor cells in arthritis 
Arthritis and rheumatism  2012;64(11):3574-3582.
Objective
To examine whether the activation of plasma kallikrein-kinin system (KKS) mediates synovial recruitment of endothelial progenitor cells (EPCs) in arthritis.
Methods
EPCs were isolated from Lewis rat bone marrow and characterized by the expression of progenitor cell lineage markers and functional property. EPCs were intravenously injected into Lewis rats bearing arthritis, their recruitment and formation of de novo blood vessels in inflamed synovium were evaluated. The role of plasma KKS was examined using a plasma kallikrein inhibitor EPI-KAL2 and an anti-kallikrein antibody 13G11. Transendothelial migration (TEM) assay was used to determine the role of bradykinin and its receptor in EPC mobilization.
Results
Lewis rat EPCs exhibited strong capacities to form tubes and vacuoles, and expressed higher level of bradykinin type 2 receptor (B2R) and progenitor cell markers CD34 and Sca-1. In Lewis rats bearing arthritis, EPCs were recruited into inflamed synovium at acute phase and formed de novo blood vessels. Inhibition of plasma kallikrein by EPI-KAL2 and 13G11 significantly suppressed synovial recruitment of EPCs and hyperproliferation of synovial cells. Bradykinin concentration-dependently stimulated TEM of EPCs, which was mediated by B2R, as the knockdown of B2R by silencing RNA completely blocked bradykinin-stimulated TEM. Moreover, bradykinin selectively upregulated the expression of homing receptor C-X-C chemokine receptor type 4 (CXCR-4) in EPCs.
Conclusion
These observations demonstrate a novel role for plasma KKS activation in the synovial recruitment of EPCs in arthritis, acting via kallirein activation and B2R-dependent mechanisms. B2R might be involved in the mobilization of EPCs via upregulation of CXCR-4.
doi:10.1002/art.34607
PMCID: PMC3477304  PMID: 22739815
21.  Salidroside exerts angiogenic and cytoprotective effects on human bone marrowderived endothelial progenitor cells via Akt/mTOR/p70S6K and MAPK signalling pathways 
British Journal of Pharmacology  2014;171(9):2440-2456.
Background and Purpose
With the increase of age, increased susceptibility to apoptosis and senescence may contribute to proliferative and functional impairment of endothelial progenitor cells (EPCs). The aim of this study was to investigate whether salidroside (SAL) can induce angiogenic differentiation and inhibit oxidative stress-induced apoptosis in bone marrow-derived EPCs (BM-EPCs), and if so, through what mechanism.
Experimental Approach
BM-EPCs were isolated and treated with different concentrations of SAL for up to 4 days. Cell proliferation, migration and tube formation ability were detected by DNA content quantification, transwell assay and Matrigel-based angiogenesis assay. Gene and protein expression were assessed by qRT-PCR and Western blot respectively.
Key Results
Treatment with SAL promoted cellular proliferation and angiogenic differentiation of BM-EPCs, and increased VEGF and NO secretion, which in turn mediated the enhanced angiogenic differentiation of BM-EPCs. Furthermore, SAL significantly attenuated hydrogen peroxide (H2O2)-induced cell apoptosis, reduced the intracellular level of reactive oxygen species and restored the mitochondrial membrane potential of BM-EPCs. Moreover, SAL stimulated the phosphorylation of Akt, mammalian target of rapamycin and p70 S6 kinase, as well as ERK1/2, which is associated with cell migration and capillary tube formation. Additionally, SAL reversed the phosphorylation of JNK and p38 MAPK induced by H2O2 and suppressed the changes in the Bax/Bcl-xL ratio observed after stimulation with H2O2.
Conclusions and Implications
These findings identify novel mechanisms that regulate EPC function and suggest that SAL has therapeutic potential as a new agent to enhance vasculogenesis as well as protect against oxidative endothelial injury.
doi:10.1111/bph.12611
PMCID: PMC3997282  PMID: 24471788
endothelial progenitor cell; salidroside; angiogenesis; reactive oxygen species; apoptosis; Akt/mTOR/p70S6K; MAPK
22.  Notch Signaling Regulates Endothelial Progenitor Cell Activity During Recovery From Arterial Injury in Hypercholesterolemic Mice 
Circulation  2010;121(9):1104-1112.
Background:
Little is known about the role of endothelial progenitor cells (EPCs) in atherosclerosis. Accordingly, we performed a series of assessments with hypercholesterolemic (ApoE−/−) and wild type (WT) mice to evaluate how cholesterol influences re-endothelialization, atherosclerosis, and EPC function after arterial injury.
Methods and Results:
Unexpectedly, re-endothelialization (assesed via resistance to Evans blue staining) and circulating EPC counts (EPC-culture assay) were greater in ApoE−/− mice than in WT mice, and transplantation of ApoE−/− bone marrow (BM) in WT mice accelerated endothelial recovery and increased recruitment of BM-derived EPCs to the neo-endothelium. Cholesterol concentration-dependently promoted the proliferation (MTS assay) of both ApoE−/− and WT EPCs, and the concentration dependence of EPC adhesion (to vitronectin-, collagen type I-, fibronectin-, and laminin-coated plates), migration (modified Boyden's-chamber assay), and anti-apoptotic (TUNEL staining) activity was biphasic. Cholesterol enhanced the mRNA expression (quantitative, real-time RT-PCR) of vascular endothelial growth factor and inhibited Notch1 mRNA expression in both ApoE−/− and WT EPCs; whereas eNOS mRNA expression increased in ApoE−/− EPCs and declined in WT EPCs after cholesterol exposure. EPC activity was greater in Notch1+/– EPCs than in WT EPCs, and transplantation of Notch1+/– BM accelerated endothelial recovery after arterial injury in WT mice.
Conclusions:
The results presented here provide novel insights into the role of EPCs during atherosclerosis and suggest that cholesterol and Notch1 may be involved in the regulation of EPC activity.
doi:10.1161/CIRCULATIONAHA.105.553917
PMCID: PMC2838724  PMID: 20176991
Atherosclerosis; Endothelial progenitor cells; Hypercholesterolemia; Notch; Nitric oxide synthase
23.  Acidic Fibroblast Growth Factor Promotes Endothelial Progenitor Cells Function via Akt/FOXO3a Pathway 
PLoS ONE  2015;10(6):e0129665.
Acidic fibroblast growth factor (FGF1) has been suggested to enhance the functional activities of endothelial progenitor cells (EPCs). The Forkhead homeobox type O transcription factors (FOXOs), a key substrate of the survival kinase Akt, play important roles in regulation of various cellular processes. We previously have shown that FOXO3a is the main subtype of FOXOs expressed in EPCs. Here, we aim to determine whether FGF1 promotes EPC function through Akt/FOXO3a pathway. Human peripheral blood derived EPCs were transduced with adenoviral vectors either expressing a non-phosphorylable, constitutively active triple mutant of FOXO3a (Ad-TM-FOXO3a) or a GFP control (Ad-GFP). FGF1 treatment improved functional activities of Ad-GFP transduced EPCs, including cell viability, proliferation, antiapoptosis, migration and tube formation, whereas these beneficial effects disappeared by Akt inhibitor pretreatment. Moreover, EPC function was declined by Ad-TM-FOXO3a transduction and failed to be attenuated even with FGF1 treatment. FGF1 upregulated phosphorylation levels of Akt and FOXO3a in Ad-GFP transduced EPCs, which were repressed by Akt inhibitor pretreatment. However, FGF1 failed to recover Ad-TM-FOXO3a transduced EPCs from dysfunction. These data indicate that FGF1 promoting EPC function is at least in part mediated through Akt/FOXO3a pathway. Our study may provide novel ideas for enhancing EPC angiogenic ability and optimizing EPC transplantation therapy in the future.
doi:10.1371/journal.pone.0129665
PMCID: PMC4463857  PMID: 26061278
24.  Reduced levels of circulating progenitor cells in juvenile idiopathic arthritis are counteracted by anti TNF-α therapy 
Background
Endothelial progenitor cells (EPC) promote angiogenesis and vascular repair. Though reduced EPC levels have been shown in rheumatoid arthritis, no study has so far evaluated EPCs in children with juvenile idiopathic arthritis (JIA). We aimed to study circulating EPCs in children with JIA, their relation to disease activity, and effects of anti TNF-α treatment.
Methods
Circulating EPCs were quantified by flow cytometry based on CD34, CD133 and KDR expression in peripheral blood of 22 patients with oligoarticular JIA and 29 age-matched controls. EPCs were re-assessed in children with methotrexate-resistant oligo-extended JIA before and up to 12 month after initiation of anti-TNF-alpha therapy. Plasma concentrations of inflammatory and EPC-regulating factors were measured using a multiplex array. Confocal immunofluorescence was used to demonstrate EPCs in synovial tissues.
Results
Children with active JIA showed a significant reduction of relative and absolute counts of circulating progenitor cells and EPCs compared to age-matched healthy controls. CD34+ cell levels were modestly and inversely correlated to disease activity. A strong inverse correlation was found between serum TNF-α and EPC levels. In 8 patients treated with anti TNF-α agents, the number of EPCs rose to values similar to healthy controls. CD34+KDR+ EPCs were found in the synovial tissue of JIA children, but not in control.
Conclusions
Children with JIA have reduced levels of the vasculoprotective and proangiogenic EPCs. While EPCs may contribute to synovial tissue remodelling, EPC pauperization may indicate an excess cardiovascular risk if projected later in life.
doi:10.1186/s12891-015-0555-9
PMCID: PMC4418050  PMID: 25925313
Stem cells; Cardiovascular; Endothelium; Angiogenesis
25.  Period 2 is essential to maintain early endothelial progenitor cell function in vitro and angiogenesis after myocardial infarction in mice 
Cellular therapeutic neovascularization has been successfully performed in clinical trials for patients with ischaemia diseases. Despite the vast knowledge of cardiovascular disease and circadian biology, the role of the circadian clock in regulating angiogenesis in myocardial infarction (MI) remains poorly understood. In this study, we aimed to investigate the role and underlying mechanisms of Period 2 (Per2) in endothelial progenitor cell (EPC) function. Flow cytometry revealed lower circulating EPC proportion in per2−/− than in wild-type (WT) mice. PER2 was abundantly expressed in early EPCs in mice. In vitro, EPCs from per2−/− mice showed impaired proliferation, migration, tube formation and adhesion. Western blot analysis demonstrated inhibited PI3k/Akt/FoxO signalling and reduced C-X-C chemokine receptor type 4 (CXCR4) protein level in EPCs of per2−/− mice. The impaired proliferation was blocked by activated PI3K/Akt/FoxO signalling. Direct interaction of CXCR4 and PER2 was detected in WT EPCs. To further study the effect of per2 on in vivo EPC survival and angiogenesis, we injected saline or DiI-labelled WT or per2−/− EPC intramyocardially into mice with induced MI. Per2−/− reduced the retention of transplanted EPCs in the myocardium, which was associated with significantly reduced DiI expression in the myocardium of MI mice. Decreased angiogenesis in the myocardium of per2−/− EPC-treated mice coincided with decreased LV function and increased infarct size in the myocardium. Per2 may be a key factor in maintaining EPC function in vitro and in therapeutic angiogenesis in vivo.
doi:10.1111/jcmm.12241
PMCID: PMC4119396  PMID: 24621388
period 2; endothelial progenitor cells; myocardial infarction; angiogenesis

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