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1.  Immunohistochemical colocalization of glycoxidation products and lipid peroxidation products in diabetic renal glomerular lesions. Implication for glycoxidative stress in the pathogenesis of diabetic nephropathy. 
Journal of Clinical Investigation  1997;100(12):2995-3004.
Advanced glycation end products (AGEs) include a variety of protein adducts whose accumulation alters the structure and function of tissue proteins and stimulates cellular responses. They have been implicated in tissue damage associated with diabetic complications. To assess the possible link between AGE accumulation and the development of diabetic nephropathy (DN), we have examined the immunohistochemical localization of various AGE structures postulated to date, i.e., pentosidine, Nepsilon-(carboxymethyl)lysine (CML), and pyrraline, in diabetic and control kidneys. CML and pentosidine accumulate in the expanded mesangial matrix and thickened glomerular capillary walls of early DN and in nodular lesions and arterial walls of advanced DN, but were absent in control kidneys. By contrast, pyrraline was not found within diabetic glomeruli but was detected in the interstitial connective tissue of both normal and diabetic kidneys. Although the distribution of pyrraline was topographically identical to type III collagen, distribution of pentosidine and CML was not specific for collagen type, suggesting that difference in matrix protein composition per se could not explain heterogeneous AGE localization. Since oxidation is linked closely to the formation of pentosidine and CML, we also immunostained malondialdehyde (MDA), a lipid peroxidation product whose formation is accelerated by oxidative stress, assuming that local oxidative stress may serve as a mechanism of pentosidine and CML accumulation. Consistent with our assumption, diabetic nodular lesions were stained positive for MDA. These findings show that AGE localization in DN varies according to AGE structure, and suggest that the colocalization of markers of glycoxidation (pentosidine and CML) with a marker of lipid peroxidation reflects a local oxidative stress in association with the pathogenesis of diabetic glomerular lesions. Thus, glycoxidation markers may serve as useful biomarkers of oxidative damage in DN.
PMCID: PMC508511  PMID: 9399945
2.  Effect of Benfotiamine on Advanced Glycation Endproducts and Markers of Endothelial Dysfunction and Inflammation in Diabetic Nephropathy 
PLoS ONE  2012;7(7):e40427.
Background
Formation of advanced glycation endproducts (AGEs), endothelial dysfunction, and low-grade inflammation are intermediate pathways of hyperglycemia-induced vascular complications. We investigated the effect of benfotiamine on markers of these pathways in patients with type 2 diabetes and nephropathy.
Methods
Patients with type 2 diabetes and urinary albumin excretion in the high-normal and microalbuminuric range (15–300 mg/24h) were randomized to receive benfotiamine (n = 39) or placebo (n = 43). Plasma and urinary AGEs (Nε-(carboxymethyl) lysine [CML], Nε-(Carboxyethyl) lysine [CEL], and 5-hydro-5-methylimidazolone [MG-H1]) and plasma markers of endothelial dysfunction (soluble vascular cell adhesion molecule-1 [sVCAM-1], soluble intercellular adhesion molecule-1 [sICAM-1], soluble E-selectin) and low-grade inflammation (high-sensitivity C-reactive protein [hs-CRP], serum amyloid-A [SAA], myeloperoxidase [MPO]) were measured at baseline and after 6 and 12 weeks.
Results
Compared to placebo, benfotiamine did not result in significant reductions in plasma or urinary AGEs or plasma markers of endothelial dysfunction and low-grade inflammation.
Conclusions
Benfotiamine for 12 weeks did not significantly affect intermediate pathways of hyperglycemia-induced vascular complications.
Trial Regristration
ClinicalTrials.gov NCT00565318
doi:10.1371/journal.pone.0040427
PMCID: PMC3391239  PMID: 22792314
3.  Advanced glycation end product deposits in climatic droplet keratopathy 
Background
Climatic droplet keratopathy (CDK), known as spheroid degeneration of the cornea, is one of the most frequent degenerative corneal disorders affecting visual function. However, the histochemical nature of the deposits seen in CDK is still unclear.
Aim
To investigate the pathogenesis of CDK, we investigated the immunohistochemical localisation of advanced glycation end products (AGEs) in surgical specimens of CDK.
Methods
Immunohistochemical localisation of Nε‐(carboxymethyl)‐L‐lysine (CML), Nε‐(carboxyethyl)‐L‐lysine (CEL), pyrraline, pentosidine and imidazolone was examined in three corneas with CDK, six corneas with bullous keratopathy and three corneas without any corneal diseases.
Results
In all the specimens with CDK, immunoreactivity was strong in CML, moderate in pyrraline and pentosidine, and weak in imidazolone. Immunoreactivity was absent in CEL. In contrast, no immunoreactivity to CML, pyrraline, pentosidine, imidazolone or CEL was detected in corneas with bullous keratopathy, or in corneas without any corneal diseases.
Conclusions
CDK is caused by an aggregation of AGE‐modified proteins. The result is consistent with etiological findings that ultraviolet irradiation and ageing, both of which are accelerators of AGE formation, are closely related to the development of CDK.
doi:10.1136/bjo.2006.099812
PMCID: PMC1857579  PMID: 16973666
4.  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
5.  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
6.  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
7.  Age-dependent increase in ortho-tyrosine and methionine sulfoxide in human skin collagen is not accelerated in diabetes. Evidence against a generalized increase in oxidative stress in diabetes. 
Journal of Clinical Investigation  1997;100(4):839-846.
The glycoxidation products Nepsilon-(carboxymethyl)lysine and pentosidine increase in skin collagen with age and at an accelerated rate in diabetes. Their age-adjusted concentrations in skin collagen are correlated with the severity of diabetic complications. To determine the relative roles of increased glycation and/or oxidation in the accelerated formation of glycoxidation products in diabetes, we measured levels of amino acid oxidation products, distinct from glycoxidative modifications of amino acids, as independent indicators of oxidative stress and damage to collagen in aging and diabetes. We show that ortho-tyrosine and methionine sulfoxide are formed in concert with Nepsilon-(carboxymethyl)lysine and pentosidine during glycoxidation of collagen in vitro, and that they also increase with age in human skin collagen. The age-adjusted levels of these oxidized amino acids in collagen was the same in diabetic and nondiabetic subjects, arguing that diabetes per se does not cause an increase in oxidative stress or damage to extracellular matrix proteins. These results provide evidence for an age-dependent increase in oxidative damage to collagen and support previous conclusions that the increase in glycoxidation products in skin collagen in diabetes can be explained by the increase in glycemia alone, without invoking a generalized, diabetes-dependent increase in oxidative stress.
PMCID: PMC508256  PMID: 9259583
8.  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
9.  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
10.  Vascular dysfunction in diabetes: The endothelial progenitor cells as new therapeutic strategy 
World Journal of Diabetes  2011;2(6):92-97.
The vascular endothelium is a critical determinant of diabetes-associated vascular complications, and improving endothelial function is an important target for therapy. Diabetes mellitus contributes to endothelial cell injury and dysfunction. Endothelial progenitor cells (EPCs) play a critical role in maintaining endothelial function and might affect the progression of vascular disease. EPCs are essential to blood vessel formation, can differentiate into mature endothelial cells, and promote the repair of damaged endothelium. In diabetes, the circulating EPC count is low and their functionality is impaired. The mechanisms that underlie this reduced count and impaired functionality are poorly understood. Knowledge of the status of EPCs is critical for assessing the health of the vascular system, and interventions that increase the number of EPCs and restore their angiogenic activity in diabetes may prove to be particularly beneficial. The present review outlines current thinking on EPCs’ therapeutic potential in endothelial dysfunction in diabetes, as well as evidence-based perspectives regarding their use for vascular regenerative medicine.
doi:10.4239/wjd.v2.i6.92
PMCID: PMC3158877  PMID: 21860692
Diabetes mellitus; Vascular dysfunction; Endothelial progenitor cells
11.  IFNα Serum Levels Are Associated with Endothelial Progenitor Cells Imbalance and Disease Features in Rheumatoid Arthritis Patients 
PLoS ONE  2014;9(1):e86069.
Introduction
IFNα has been largely implicated in the ethiopathogenesis of autoimmune diseases but only recently it has been linked to endothelial damage and accelerated atherosclerosis in autoimmunity. In addition, proinflammatory conditions are supposed to be implicated in the cardiovascular status of these patients. Since a role for IFNα in endothelial damage and impaired Endothelial Progenitor Cell (EPC) number and function has been reported in other diseases, we aimed to evaluate the potential associations of IFNα serum levels on EPC populations and cytokine profiles in Rheumatoid Arthritis (RA) patients.
Methods
pre-EPC, EPC and mature EPC (mEPC) populations were quantified by flow cytometry analyzing their differential CD34, CD133 and VEGFR2 expression in blood samples from 120 RA patients, 52 healthy controls (HC), and 83 systemic lupus erythematosus (SLE) patients as disease control. Cytokine serum levels were measured by immunoassays and clinical and immunological data, including cardiovascular (CV) events and CV risk factors, were retrospectively obtained by reviewing clinical records.
Results
Long-standing, but not recent onset RA patients displayed a significant depletion of all endothelial progenitor populations, unless high IFNα levels were present. In fact, the IFNhigh RA patient group (n = 40, 33%), showed increased EPC levels, comparable to SLE patients. In addition, high IFNα serum levels were associated with higher disease activity (DAS28), presence of autoantibodies, higher levels of IL-1β, IL-6, IL-10 and MIP-1α, lower amounts of TGF-β, and increased mEPC/EPC ratio, thus suggesting higher rates of endothelial damage and an endothelial repair failure. Finally, the relationship between high IFNα levels and occurrence of CV events observed in RA patients seems to support this hypothesis.
Conclusions
IFNα serum marker could be used to identify a group of RA patients with increased disease activity, EPC imbalance, enhanced proinflammatory profile and higher cardiovascular risk, probably due, at least in part, to an impaired endothelial repair.
doi:10.1371/journal.pone.0086069
PMCID: PMC3897639  PMID: 24465874
12.  Endothelial progenitor cell transplantation improves long-term outcome in mice 
Annals of neurology  2010;67(4):488-497.
Endothelial progenitor cells (EPCs) have been implicated in playing an important role in vascular repair and revascularization in ischemic organs including brain tissue. However, the cause of EPC migration and the function of EPC playing following post-ischemia are unclear. Here, we reported EPC therapy in a mouse model of transient middle cerebral artery occlusion (tMCAO) to explore the roles of EPC following ischemic brain injury.
Human EPCs were cultured, characterized, and confirmed with flow cytometry. Ex vivo expanded EPCs (1×106) were injected via jugular vein after 1 hour of tMCAO. Histological and behavioral analyses were performed from day 1 to 28 days after tMCAO.
EPCs were detected in ischemic brain region 24 hours after MCAO. EPC transplantation significantly reduced ischemic infarct volume at 3 days following MCAO compared to the control (p<0.05). CXCR4 was expressed on majority of EPCs and SDF-1-induced EPC migration was blocked by AMD3100 in vitro. SDF-1 was up-regulated in ischemic brain and AMD3100 could reduce EPCs migration to the ischemic region in vivo, suggesting that SDF-1/CXCR4 was involved in EPC-mediated neuroprotection. Compared to the control, EPC therapy reduced mouse cortex atrophy 4 weeks after tMCAO, which was accompanied by improved neurobehavioral outcomes (p<0.05). In addition, EPC injection potently increased angiogenesis in the peri-infarction area (p<0.05).
We conclude that systemic delivery of EPC protect against cerebral ischemic injury, promote neurovascular repair, and improve long-term neurobehavioral outcomes. Our data suggests that SDF-1/CXCR4 plays a critical role in EPC-mediated neuroprotection.
doi:10.1002/ana.21919
PMCID: PMC3026588  PMID: 20437584
angiogenesis; EPCs; ischemia; mice; neuroprotection
13.  Circulating endothelial and progenitor cells: Evidence from acute and long-term exercise effects 
World Journal of Cardiology  2012;4(12):312-326.
Circulating bone-marrow-derived cells, named endothelial progenitor cells (EPCs), are capable of maintaining, generating, and replacing terminally differentiated cells within their own specific tissue as a consequence of physiological cell turnover or tissue damage due to injury. Endothelium maintenance and restoration of normal endothelial cell function is guaranteed by a complex physiological procedure in which EPCs play a significant role. Decreased number of peripheral blood EPCs has been associated with endothelial dysfunction and high cardiovascular risk. In this review, we initially report current knowledge with regard to the role of EPCs in healthy subjects and the clinical value of EPCs in different disease populations such as arterial hypertension, obstructive sleep-apnea syndrome, obesity, diabetes mellitus, peripheral arterial disease, coronary artery disease, pulmonary hypertension, and heart failure. Recent studies have introduced the novel concept that physical activity, either performed as a single exercise session or performed as part of an exercise training program, results in a significant increase of circulating EPCs. In the second part of this review we provide preliminary evidence from recent studies investigating the effects of acute and long-term exercise in healthy subjects and athletes as well as in disease populations.
doi:10.4330/wjc.v4.i12.312
PMCID: PMC3530787  PMID: 23272272
Circulating endothelial cells; Circulating progenitor cells; Exercise; Cardiovascular disease
14.  Citric acid inhibits development of cataracts, proteinuria and ketosis in streptozotocin (type1) diabetic rats 
Although many fruits such as lemon and orange contain citric acid, little is known about beneficial effects of citric acid on health. Here we measured the effect of citric acid on the pathogenesis of diabetic complications in streptozotocin-induced diabetic rats. Although oral administration of citric acid to diabetic rats did not affect blood glucose concentration, it delayed the development of cataracts, inhibited accumulation of advanced glycation end products (AGEs) such as Nε-(carboxyethyl)lysine (CEL) and Nε-(carboxymethyl)lysine (CML) in lens proteins, and protected against albuminuria and ketosis . We also show that incubation of protein with acetol, a metabolite formed from acetone by acetone monooxygenase, generate CEL, suggesting that inhibition of ketosis by citric acid may lead to the decrease in CEL in lens proteins. These results demonstrate that the oral administration of citric acid ameliorates ketosis and protects against the development of diabetic complications in an animal model of type 1 diabetes.
doi:10.1016/j.bbrc.2010.01.095
PMCID: PMC2917331  PMID: 20117096
Advanced glycation end-product (AGEs); Nε-(carboxyethyl)lysine (CEL); cataract; diabetes; ketosis; nephropathy
15.  Endothelial Colony Forming Cell role in neoangiogenesis and tissue repair 
Purpose of review
Patients suffering from vascular disease often have impaired angiogenic ability contributing to impaired tissue repair. One potential therapy is to deliver cells that can aid in angiogenesis. This review will discuss the ability of endothelial progenitor cells (EPC), which have been reported to contribute to neoangiogenesis in both physiological and pathological conditions, to contribute to neoangiogenesis in tissue repair.
Recent findings
In recent years, various reports have described conflicting roles for EPC in vessel formation. Currently there are three different assays for outgrowth of EPC all resulting in the isolation of different cell populations. This confusion is partially due to limited functional characterization of putative EPC populations. One population, ECFC, have been shown to possess all the characteristics of a true endothelial progenitor.
Summary
The review overviews the role of putative EPC populations in angiogenesis and tissue repair. While all EPC populations have been shown to play a role in angiogenesis, only ECFC have demonstrated the ability to form de novo blood vessels in vivo. Additionally ECFC have been shown to play a role in neovascularization in several pre-clinical rodent models suggesting the may be an excellent cell source for treatment of patients with diminished vascular function.
doi:10.1097/MOT.0b013e32833454b5
PMCID: PMC2880951  PMID: 19898235
endothelial progenitor cell; endothelial colony forming cell; angiogenesis; wound healing
16.  Phloroglucinol Inhibits the in vitro Differentiation Potential of CD34 Positive Cells into Endothelial Progenitor Cells 
Biomolecules & Therapeutics  2012;20(2):158-164.
Inhibiting the bioactivities of circulating endothelial progenitor cells (EPCs) results in significant inhibition of neovessel formation during tumor angiogenesis. To investigate the potential effect of phloroglucinol as an EPC inhibitor, we performed several in vitro functional assays using CD34+ cells isolated from human umbilical cord blood (HUCB). Although a high treatment dose of phloroglucinol did not show any cell toxicity, it specifically induced the cell death of EPCs under serum free conditions through apoptosis. In the EPC colony-forming assay (EPC-CFA), we observed a significant decreased in the small EPC-CFUs for the phloroglucinol group, implying that phloroglucinol inhibited the early stage of EPC commitment. In addition, in the in vitro expansion assay using CD34+ cells, treatment with phloroglucinol was shown to inhibit endothelial lineage commitment, as demonstrated by the decrease in endothelial surface markers of EPCs including CD34+, CD34+/CD133+, CD34+/CD31+ and CD34+/CXCR4+. This is the first report to demonstrate that phloroglucinol can inhibit the functional bioactivities of EPCs, indicating that phloroglucinol may be used as an EPC inhibitor in the development of biosafe anti-tumor drugs that target tumor angiogenesis.
doi:10.4062/biomolther.2012.20.2.158
PMCID: PMC3792212  PMID: 24116289
Endothelial progenitor cell; Tumor angiogenesis; Phloroglucinol; Colony forming assay
17.  Vascular stem cells in diabetic complications: evidence for a role in the pathogenesis and the therapeutic promise 
Long standing diabetes leads to structural and functional alterations in both the micro- and the macro-vasculature. Vascular endothelial cells (ECs) are the primary target of the hyperglycemia-induced adverse effects. Vascular stem cells that give rise to endothelial progenitor cells (EPCs) and mesenchymal progenitor cells (MPCs) represent an attractive target for cell therapy for diabetic patients. A number of studies have reported EPC dysfunction as a novel participant in the culmination of the diabetic complications. The controversy behind the identity of EPCs and the similarity between these progenitor cells to hematopoietic cells has led to conflicting results. MPCs, on the other hand, have not been examined for a potential role in the pathogenesis of the complications. These multipotent cells, however, do show a therapeutic role. In this article, we summarize the vascular changes that occur in diabetic complications highlighting some of the common features, the key findings that illustrate an important role of vascular stem cells (VSCs) in the pathogenesis of chronic diabetic complications, and provide mechanisms by which these cells can be used for therapy.
doi:10.1186/1475-2840-11-37
PMCID: PMC3476432  PMID: 22524626
Diabetes; Diabetic complications; Angiopathy; Endothelial cells; Vasculogenesis; Angiogenesis; Stem cells; Progenitors; Perivascular cells
18.  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
19.  Comparison of endothelial progenitor cell function in type 2 diabetes with good and poor glycemic control 
Background
Endothelial progenitor cells (EPCs) play an important role in vascular repair and a decrease in the number of EPCs is observed in type 2 diabetes. However, there is no report on the change of EPCs after glycemic control. This study therefore aimed to investigate the EPC number and function in patients with good and poor glycemic control.
Methods
The number of EPCs was studied using flow cytometry by co-expression of CD34 and VEGFR2. The EPCs were cultured and characterized by the expression of UEA-I, CD34, VEGFR2, vWF and Dil-Ac-LDL engulfment, as well as the ability to form capillary-like structures. An in vitro study on the effect of hyperglycemia on the proliferation and viability of the cultured EPCs was also performed.
Results
The number of EPCs in type 2 diabetes was significantly decreased compared with healthy controls and there was an inverse correlation between the EPC numbers and plasma glucose, as well as HbA1C. The number and function of EPCs in patients with good glycemic control were recovered compared with those with poor glycemic control. When glucose was supplemented in the culture in vitro, there was a negative effect on the proliferation and viability of EPCs, in a dose-dependent manner, whereas the enhancement of apoptosis was observed.
Conclusion
There was EPC dysfunction in type 2 diabetes which might be improved by strict glycemic control. However, the circulating EPC number and proliferative function in patients with good glycemic control did not reach the level in healthy controls.
doi:10.1186/1472-6823-10-5
PMCID: PMC2858721  PMID: 20374643
20.  Effects of Endothelial Progenitor Cell-Derived Microvesicles on Hypoxia/Reoxygenation-Induced Endothelial Dysfunction and Apoptosis 
Oxidative stress-induced endothelial dysfunction plays a key role in ischemia/reperfusion injury. Recent evidence indicates that endothelial progenitor cell-derived microvesicles (EPC-MVs) can promote angiogenesis of endothelial cells (ECs). Here, we investigated the potential effects of EPC-MVs on hypoxia/reoxygenation (H/R) injury in human brain microvascular ECs (hb-ECs). MVs were prepared from EPCs cultured in a serum deprivation (SD) medium (starving stress, sEPC-MVs) or SD medium containing tumor necrosis factor-α (TNFα) (apoptotic stress, aEPC-MVs). H/R injury model of hb-ECs was produced by 6 hr hypoxia (1% O2) and 24 hr reoxygenation. The H/R hb-ECs were co-cultured with EPC-MVs. Results showed that (1) H/R hb-ECs were dysfunctional and coupled with increased apoptosis and ROS overproduction; (2) under two different conditions, EPCs displayed remarkable difference in caspase 3 and miR126 expression, which were carried by the corresponsive EPC-MVs; (3) functionally, sEPC-MVs had beneficial effects on H/R hb-ECs, whereas aEPC-MVs had detrimental effects; (4) the diverse effects of sEPC-MVs and aEPC-MVs were associated with the changes in miR126 and eNOS expression and were abolished by PI3K inhibitor. In conclusion, sEPCs-MVs and aEPC-MVs are functionally different on hb-EC apoptosis and dysfunction via their carried RNAs associated with ROS production and PI3K/eNOS/NO pathway.
doi:10.1155/2013/572729
PMCID: PMC3830832  PMID: 24288585
21.  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
22.  Oxidative Stress Impairs Endothelial Progenitor Cell Function 
Antioxidants & redox signaling  2008;10(11):1895-1907.
Circulating endothelial progenitor cells (EPCs) in adult human peripheral blood were identified in 1997. Since their original identification, EPCs have been extensively studied as biomarkers to assess the risk of cardiovascular disease in human subjects and as a potential cell therapeutic for vascular regeneration. EPCs are exposed to oxidative stress during vascular injury as residents of blood vessel walls or as circulating cells homing to sites of neovascularization. Given the links between oxidative injury, endothelial cell dysfunction, and vascular disease, recent investigation has focused on the responses of EPCs to oxidant stress and the molecular mechanisms, which control redox regulation in these specialized cells. In this review we discuss the various cell and flow cytometric techniques used to define and isolate EPCs from circulating blood and the current human and mouse genetic data, which offer insights into redox control in EPC biology and angiogenesis. Finally, we review how EPC responses to oxidant stress may be a critical determinant in maintaining the integrity and function of the cardiovascular system and how perturbations of redox control in EPCs may lead to various human diseases.
doi:10.1089/ars.2008.2118
PMCID: PMC2575029  PMID: 18627346
23.  Oxidative Stress Impairs Endothelial Progenitor Cell Function 
Antioxidants & Redox Signaling  2008;10(11):1895-1907.
Abstract
Circulating endothelial progenitor cells (EPCs) in adult human peripheral blood were identified in 1997. Since their original identification, EPCs have been extensively studied as biomarkers to assess the risk of cardiovascular disease in human subjects and as a potential cell therapeutic for vascular regeneration. EPCs are exposed to oxidative stress during vascular injury as residents of blood vessel walls or as circulating cells homing to sites of neovascularization. Given the links between oxidative injury, endothelial cell dysfunction, and vascular disease, recent investigation has focused on the responses of EPCs to oxidant stress and the molecular mechanisms that control redox regulation in these specialized cells. In this review, we discuss the various cell and flow-cytometric techniques used to define and isolate EPCs from circulating blood and the current human and mouse genetic data, which offer insights into redox control in EPC biology and angiogenesis. Finally, we review how EPC responses to oxidant stress may be a critical determinant in maintaining the integrity and function of the cardiovascular system and how perturbations of redox control in EPCs may lead to various human diseases. Antioxid. Redox Signal. 10, 1895–1907.
doi:10.1089/ars.2008.2118
PMCID: PMC2575029  PMID: 18627346
24.  Endothelial progenitor cell: A blood cell by many other names may serve similar functions 
The field of endothelial progenitor cell (EPC) biology is approaching a decade and a half since generating substantial promise as a potential reparative cell therapy for a spectrum of human clinical disorders. With considerable speed, scientists and clinicians moved from basic studies of isolating and characterizing the biologic properties of EPCs, to pre-clinical EPC treatment studies in rodent model systems of cardiovascular disease, and to the delivery of EPC or marrow-derived cells into selected human subjects (reviewed in [1, 2]). In some disease settings, patient benefits from the infused EPC or marrow-derived cells have been documented, though perhaps not to the extent hoped for or predicted by the results in the preclinical animal model systems [3]. In most human clinical trials, autologous bone marrow mononuclear cells have been infused into patients with cardiovascular disease in an attempt to provide certain presumed EPC subsets to ameliorate ischemic insult [4-7]. To provide some perspective on the advances to date, this review will begin by highlighting the major clarifications in EPC definitions that have occurred over the past 10 years and how this information has instructed changes to the selection of bone marrow subsets for patient use [8-11]. To bring perspective to the increased appreciation of the roles played by hematopoietic cells in vascular repair, we will provide an overview of the hematopoietic hierarchy in mouse and man and identify those subsets that display proangiogenic activities. This perspective may help the reader consider crucial milestones in the discovery and application of HSC and progenitor cells as a cell therapeutic that have not been well explored in the EPC field. The review will conclude with a list of issues that need to be addressed to permit a more quantitative and definable nomenclature for the cells that participate in vascular endothelial repair and replacement. This review will not address the role of those EPC comprised of resident or circulating endothelial cells or endothelial colony forming cells involved in vascular repair and regeneration under normal or pathological conditions (reviewed in [8-15]).
doi:10.1007/s00109-013-1002-8
PMCID: PMC3704045  PMID: 23371317
25.  The possible role of ribosomal protein S6 kinase 4 in the senescence of endothelial progenitor cells in diabetes mellitus 
Background
The decrease and dysfunction of endothelial progenitor cells (EPCs) has been assumed as an important cause/consequence of diabetes mellitus (DM) and its complications, in which the senescence of EPCs induced by hyperglycemia may play an immensurable role. However, the mechanisms of EPCs senescence has not been fully investigated. Recently, ribosomal protein S6 kinase 4 (RSK4), a member of serine/threomine (Ser/Thr) kinase family and p53-related gene, is reported to regulate the replicative and stress-induced senescence of different cells.
Presentation of the hypothesis
These above lead to consideration of an evidence-based hypothesis that RSK4 may serve as a mediator of EPCs senescence in DM.
Testing the hypothesis
EPCs of healthy subjects and DM patients are isolated from peripheral blood and incubated with high glucose (HG). Then, the EPCs senescence would be detected by senescence associated β-galactosides (SA-β-gal) staining. Meanwhile, the RSK4 expression is assessed by RT-PCR and western blot. Moreover, overexpressing or RNA interfering of RSK4 in EPCs to investigate the relationship between RSK4 expression and the senescence of EPCs are necessary to substantiate this hypothesis. Also, studies on possible upstream and downstream factors of RSK4 would be explored to reveal the RSK4-mediated senescence pathway in EPCs.
Implications of the hypothesis
If proved, this hypothesis will provide another mediator of EPCs senescence, and may establish a novel pathogenesis for DM and further benefit to the management of DM.
doi:10.1186/1475-2840-11-12
PMCID: PMC3295662  PMID: 22297070
Diabetes mellitus; Endothelial progenitor cell; RSK4; Senescence

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