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.
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.
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.
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.
Benfotiamine for 12 weeks did not significantly affect intermediate pathways of hyperglycemia-induced vascular complications.
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.
To investigate the pathogenesis of CDK, we investigated the immunohistochemical localisation of advanced glycation end products (AGEs) in surgical specimens of CDK.
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.
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.
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.
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.
advanced glycation end-products; Nε-(carboxymethyl)lysine; Nε-(carboxyethyl)lysine; epidemiology; blood sampling
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.
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.
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.
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.
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.
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.
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.
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.
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.
Endothelial progenitor cell; Tumor angiogenesis; Phloroglucinol; Colony forming assay
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.
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.
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.
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.
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.
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.
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.
Endothelial progenitor cells; AGEs; Diabetes; Vasoactive substances
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.
Diabetes mellitus; Vascular dysfunction; Endothelial progenitor cells
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.
Advanced glycation end-product (AGEs); Nε-(carboxyethyl)lysine (CEL); cataract; diabetes; ketosis; nephropathy
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.
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.
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.
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.
Intracoronary delivery of endothelial progenitor cells (EPCs) is an emerging concept for the treatment of cardiovascular disease. Enhancement of EPC adhesion to vascular endothelium could improve cell retention within targeted organs. Because extracellular adenosine is elevated at sites of ischemia and stimulates neovascularization, we examined the potential role of adenosine in augmenting EPC retention to cardiac microvascular endothelium. Stimulation of adenosine receptors in murine embryonic EPCs (eEPCs) and cardiac endothelial cells (cECs) rapidly, within minutes, increased eEPC adhesion to cECs under static and flow conditions. Similarly, adhesion of human adult culture-expanded EPCs to human cECs was increased by stimulation of adenosine receptors. Furthermore, adenosine increased eEPC retention in isolated mouse hearts perfused with eEPCs. We determined that eEPCs and cECs preferentially express functional A1 and A2B adenosine receptor subtypes, respectively, and that both subtypes are involved in the regulation of eEPC adhesion to cECs. We documented that the interaction between P-selectin and its ligand (P-selectin glycoprotein ligand-1) plays a role in adenosine-dependent eEPC adhesion to cECs and that stimulation of adenosine receptors in cECs induces rapid cell surface expression of P-selectin. Our results suggest a role for adenosine in vasculogenesis and its potential use to stimulate engraftment in cell-based therapies.
adenosine; adenosine receptors; endothelium; adhesion molecules
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.
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.
Diverse subsets of endothelial progenitor cells (EPCs) are used for the treatment of ischemic diseases in clinical trials and circulating EPCs levels are considered as biomarkers for coronary and peripheral artery disease. However, despite significant steps forward in defining their potential for both therapeutic and diagnostic purposes, further progress has been mined by unresolved questions around the definition and the mechanism of action of EPCs. Diverse culturing methods and detection of various combinations of different surface antigens were used to enrich and identify EPCs. These attempts were particularly challenged by the close relationship and overlapping markers of the endothelial and hematopoietic lineages. This article will critically review the most commonly used protocols to define EPCs by culture assays or by FACS in the context of their therapeutic or diagnostic use. We also delineate new research avenues to move forward our knowledge on EPC biology.
progenitor cells; stem cells; angiogenesis; endothelial progenitor cells; risk factor
Bone marrow (BM)-derived endothelial progenitor cells (EPC) have therapeutic potentials in promoting tissue regeneration, but how these cells are modulated in vivo has been elusive. Here, we report that RBP-J, the critical transcription factor mediating Notch signaling, modulates EPC through CXCR4. In a mouse partial hepatectomy (PHx) model, RBP-J deficient EPC showed attenuated capacities of homing and facilitating liver regeneration. In resting mice, the conditional deletion of RBP-J led to a decrease of BM EPC, with a concomitant increase of EPC in the peripheral blood. This was accompanied by a down-regulation of CXCR4 on EPC in BM, although CXCR4 expression on EPC in the circulation was up-regulated in the absence of RBP-J. PHx in RBP-J deficient mice induced stronger EPC mobilization. In vitro, RBP-J deficient EPC showed lowered capacities of adhering, migrating, and forming vessel-like structures in three-dimensional cultures. Over-expression of CXCR4 could at least rescue the defects in vessel formation by the RBP-J deficient EPC. These data suggested that the RBP-J-mediated Notch signaling regulated EPC mobilization and function, at least partially through dynamic modulation of CXCR4 expression. Our findings not only provide new insights into the regulation of EPC, but also have implications for clinical therapies using EPC in diseases.
Endothelial progenitor cells (EPCs) are involved in the maintenance of endothelial homoeostasis and in the process of new vessel formation. Experimental and clinical studies have shown that atherosclerosis is associated with reduced numbers and dysfunction of EPCs; and that medications alone are able to partially reverse the impairment of EPCs in patients with atherosclerosis. Therefore, novel EPC-based therapies may provide enhancement in restoring EPCs’ population and improvement of vascular function. Here, for a better understanding of the molecular mechanisms underlying EPC impairment in atherosclerosis, we provide a comprehensive overview on EPC characteristics, phenotypes, and the signaling pathways underlying EPC impairment in atherosclerosis.
Endothelial progenitor cells; Atherosclerosis; Inflammation; Review
The identification of endothelial progenitor cells (EPCs) has led to a significant paradigm in the field of vascular biology and opened a door to the development of new therapeutic approaches. Based on the current evidence, it appears that EPCs may make both direct contribution to neovascularization and indirectly promote the angiogenic function of local endothelial cells via secretion of angiogenic factors. This concept of arterial wall repair mediated by bone marrow (BM)-derived EPCs provided an alternative to the local “response to injury hypothesis” for development of atherosclerotic inflammation. Increased oxidant stress has been proposed as a molecular mechanism for endothelial dysfunction, in part by reducing nitric oxide (NO) bioavailability. EPCs function may also be highly dependent on a well-controlled oxidant stress because EPCs NO bioavailability (which is highly sensitive to oxidant stress) is critical for their in vivo function. The critical question is whether oxidant damage directly leads to an impairment in EPCs function. It was revealed that activation of angiotensin II (Ang II) type 1 receptor stimulates nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase in the vascular endothelium and leads to production of reactive oxygen species. We observed that Ang II accelerates both BM- and peripheral blood (PB)-derived EPCs senescence by a gp91phox-mediated increase of oxidative stress, resulting in EPCs dysfunction. Consistently, both Ang II receptor 1 blockers (ARBs) and angiotensin converting enzyme (ACE) inhibitors have been reported to increase the number of EPCs in patients with cardiovascular disease. In this review, we describe current understanding of the contributions of oxidative stress in cardiovascular disease, focusing on the potential mechanisms of EPCs senescence.
Endothelial progenitor cell; oxidative stress; senescence; angiotensin II; telomerase; nitric oxide.
It has been recently demonstrated that endothelial progenitor cells (EPCs) have increasing potential for gene therapy or regenerative cell therapy for cardiovascular diseases and cancer. However, current therapies involving EPCs are inefficient because of the very low level of EPCs in the available sources, for example, in blood. One solution is to derive in vitro an expanded population of EPCs from circulation. In addition, EPCs like other progenitor cells have an intrinsic predisposition of differentiating into mature cell types, for example, mature endothelial cells; therefore, establishing a sufficient amount of EPCs alongside maintaining the EPC characteristic phenotype during genetic modification and long-term culture presents a significant challenge to the field of gene and cell therapies. In this study, we have systematically investigated EPCs from different sources and used multiple parameters, including cell surface markers and a tubule formation assay to identify factors that influence the establishment, characteristics, and vector transduction capability of EPCs. Our results show the considerable promise, as well as certain limitations in the establishment and manipulation of genetically modified EPCs for gene therapy. While obtaining high transduction efficiency and robust in vitro tubule formation of EPCs using lentiviral vectors, we also observed that lentiviral vector transduction significantly altered EPC phenotype as demonstrated by an increased percentage of CD34+ progenitor cells and increased expression of adhesion molecule CD144 (VE-cadherin). Taking account of the increased expression of CD144 reported in cancer patients, the altered expression of EPC-related markers, for example, VE-cadherin and the enrichment of CD34+ cells, after vector transduction indicates the importance of extensive characterization and vigorous safety control of genetically modified EPCs before they are accepted for clinical use.
Werling and colleagues systematically profile endothelial progenitor cells (EPC) from different sources in order to identify factors that influence EPC establishment and vector transduction capability. While lentiviral vector transduction resulted in high transduction efficiency and robust in vitro tubule formation of EPC, it also significantly altered EPC phenotype. Therefore, extensive characterization and vigorous safety control of genetically modified EPC is warranted before clinical use.
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.
Circulating endothelial cells; Circulating progenitor cells; Exercise; Cardiovascular disease
The effects of anaerobic (lens) vs aerobic (skin) environment on carbonyl and oxidant stress are compared using de novo and existing data on advanced glycation and oxidation products in human crystallins and collagen. Almost all modifications increase with age. Methylglyoxal hydroimidazolones (MG-H1), carboxymethyl-lysine (CML), and carboxyethyl-lysine (CEL) are several folds higher in lens than skin, and markedly increase upon incubation of lens crystallins with 5 mM ascorbic acid. Vice-versa, fructose-lysine, glucosepane crosslinks, glyoxal hydroimidazolones (G-H1), metal catalyzed oxidation (allysine) and H2O2 dependent modifications (2-aminoapidic acid and methionine sulfoxide) are markedly elevated in skin, but relatively suppressed in the aging lens. In both tissues ornithine is the dominant modification, implicating arginine residues as the principal target of the Maillard reaction in vivo. Diabetes (here mostly type 2 studied) increases significantly fructose-lysine and glucosepane in both tissues (P<0.001) but has surprisingly little effect on the absolute level of most other advanced glycation end products (AGEs) . However, diabetes strengthens the Spearman correlation coefficients for age-related accumulation of hydrogen peroxide mediated modifications in the lens. Overall, the data suggest oxoaldehyde stress involving methylglyoxal from either glucose or ascorbate is predominant in the aging non-cataractous lens, while aging skin collagen undergoes combined attack by non-oxidative glucose mediated modifications, as well as those from metal catalyzed oxidation and H2O2.
crystallins; collagen; glycation; oxidative stress; methylglyoxal; metals
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.
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.
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.
endothelial progenitor cell; endothelial colony forming cell; angiogenesis; wound healing
Cell-based therapy has emerged as a promising therapeutic tool for treatment of ischemic cardiovascular disease. Both unselected bone marrow-derived mononuclear cells (BMNCs), which include stem/progenitor cells and several other cell types, and endothelial progenitor cells (EPCs), a subpopulation of BMNCs, display regenerative potential in ischemic tissue. Abundant evidence supports the involvement of EPCs in capillary growth, and EPCs also appear to participate in the formation of collateral vessels. Collectively, these effects have led to improved perfusion and functional recovery in animal models of myocardial and peripheral ischemia, and in early clinical trials, the therapeutic administration of EPCs to patients with myocardial infarction or chronic angina have been associated with positive trends in perfusion. EPCs also contribute to endothelial repair and may, consequently, impede the development or progression of arteriosclerosis. This review provides a brief summary of the preclinical and clinical evidence for the role of EPCs in blood-vessel formation and repair during ischemic cardiovascular disease.
Endothelial progenitor cell; Vasculogenesis; Collateral formation