Concomitant overexpression of triple antioxidant enzymes selectively increases circulating endothelial progenitor cells in mice with limb ischaemia

Endothelial progenitor cells (EPCs) are a group of heterogeneous cells in bone marrow (BM) and blood. Ischaemia increases reactive oxygen species (ROS) production that regulates EPC number and function. The present study was conducted to determine if ischaemia‐induced ROS differentially regulated individual EPC subpopulations using a mouse model concomitantly overexpressing superoxide dismutase (SOD)1, SOD3 and glutathione peroxidase. Limb ischaemia was induced by femoral artery ligation in male transgenic mice with their wild‐type littermate as control. BM and blood cells were collected for EPCs analysis and mononuclear cell intracellular ROS production, apoptosis and proliferation at baseline, day 3 and day 21 after ischaemia. Cells positive for c‐Kit⁺/CD31⁺ or Sca‐1⁺/Flk‐1⁺ or CD34⁺/CD133⁺ or CD34⁺/Flk‐1⁺ were identified as EPCs. ischaemia significantly increased ROS production and cell apoptosis and decreased proliferation of circulating and BM mononuclear cells and increased BM and circulating EPCs levels. Overexpression of triple antioxidant enzymes effectively prevented ischaemia‐induced ROS production with significantly decreased cell apoptosis and preserved proliferation and significantly increased circulating EPCs level without significant changes in BM EPC populations, associated with enhanced recovery of blood flow and function of the ischemic limb. These data suggested that ischaemia‐induced ROS was differentially involved in the regulation of circulating EPC population.     

Ambient Fine Particulate Matter Suppresses In Vivo Proliferation of Bone Marrow Stem Cells through Reactive Oxygen Species Formation

Aims

Some environmental insults, such as fine particulate matter (PM) exposure, significantly impair the function of stem cells. However, it is unknown if PM exposure could affect the population of bone marrow stem cells (BMSCs). The present study was to investigate the effects of PM on BMSCs population and related mechanism(s).

Main Metheods

PM was intranasally distilled into male C57BL/6 mice for one month. Flow cytometry with antibodies for BMSCs, Annexin V and BrdU ware used to determine the number of BMSCs and the levels of their apoptosis and proliferation in vivo. Phosphorylated Akt (P-Akt) level was determined in the BM cells with western blotting. Intracellular reactive oxygen species (ROS) formation was quantified using flow cytometry analysis. To determine the role of PM-induced ROS in BMSCs population, proliferation, and apotosis, experiments were repeated using N-acetylcysteine (NAC)-treated wild type mice or a triple transgenic mouse line with overexpression of antioxidant network (AON) composed of superoxide dismutase (SOD)1, SOD3, and glutathione peroxidase-1 with decreased in vivo ROS production.

Key Findings

PM treatment significantly reduced BMSCs population in association with increased ROS formation, decreased P-Akt level, and inhibition of proliferation of BMSCs without induction of apoptosis. NAC treatment or AON overexpression with reduced ROS formation effectively prevented PM-induced reduction of BMSCs population and proliferation with partial recovery of P-Akt level.

Significance

PM exposure significantly decreased the population of BMSCs due to diminished proliferation via ROS-mediated mechanism (could be partially via inhibition of Akt signaling).     

内皮祖细胞在炎症损伤修复中的作用和机制

内皮祖细胞（endothelial progenitor cells,EPCs）是出生后,可以在机体内分化为成熟内皮细胞的一种前体细胞,主要来源于骨髓。多种伴有血管内皮细胞损伤的疾病都可引起外周血EPCs数量变化。有研究显示EPCs参与炎性损伤修复,并且外周血EPCs数量与血管内皮损伤程度和疾病预后存在一定的相关关系。EPCs。通过动员、迁移、归巢和分化等步骤修复内皮。炎症反应中受损组织释放的基质细胞衍生因子、血管内皮生长因子可与EPCs相应的受体结合,通过内皮型一氧化氮合酶、基质金属蛋白酶9等途径调节内皮修复过程,这是EPCs分化为内皮细胞过程的主要调控机制。此外,EPCs还可通过旁分泌机制促进相邻的内皮细胞增殖分化。目前,EPCs在炎症领域仅用于内皮炎性损伤和疾病预后评估,但是EPCs在心血管疾病和组织工程领域应用研究的成功,为EPCs在炎症反应的诊断和治疗提供了新的思路。     

Angiotensin II promotes NO production, inhibits apoptosis and enhances adhesion potential of bone marrow-derived endothelial progenitor cells

Endothelial progenitor cells (EPCs) participate in the processes of postnatal neovascularization and re-endothelialization in response to tissue ischemia and endothelial injury. The level of EPCs present has been found to be directly associated with the outcome of cardiovascular diseases, and could be regulated by stimulatory or inhibitory factors. Given the close relationship between angiotensin II (AngII) and the cardiovascular system, we investigated the effect of AngII on the activities of bone marrow (BM)-derived EPCs. Cells were isolated from BM of rats by density gradient centrifugation. Administration of AngII significantly promoted nitric oxide (NO) release, inhibited EPC apoptosis and enhanced EPC adhesion potential. All of these AngII-mediated effects on EPCs were attenuated by pretreatment with valsartan or L-NAME. Moreover, both LY294002 and wortmannin abolished the anti-apoptotic effect of AngII. Western blot analyses indicated that endothelial NO synthase (eNOS) protein and phosphorylated Akt increased with the treatment of AngII in EPCs. Thus, AngII improved several activities of EPCs through AngII type 1 receptor (AT1R), which may represent a possible mechanism linking AngII and AT1R with angiogenesis. Additionally, AngII-induced NO synthesis through eNOS in EPCs regulates apoptosis and adhesion, and the PI3-kinase/Akt pathway has an essential role in AngII-induced antiapoptosis signaling.     

Redox regulation of stem/progenitor cells and bone marrow niche

Bone marrow (BM)-derived stem and progenitor cell functions including self-renewal, differentiation, survival, migration, proliferation, and mobilization are regulated by unique cell-intrinsic and -extrinsic signals provided by their microenvironment, also termed the “niche.” Reactive oxygen species (ROS), especially hydrogen peroxide (H₂O₂), play important roles in regulating stem and progenitor cell functions in various physiologic and pathologic responses. The low level of H₂O₂ in quiescent hematopoietic stem cells (HSCs) contributes to maintaining their “stemness,” whereas a higher level of H₂O₂ within HSCs or their niche promotes differentiation, proliferation, migration, and survival of HSCs or stem/progenitor cells. Major sources of ROS are NADPH oxidase and mitochondria. In response to ischemic injury, ROS derived from NADPH oxidase are increased in the BM microenvironment, which is required for hypoxia and hypoxia-inducible factor-1α expression and expansion throughout the BM. This, in turn, promotes progenitor cell expansion and mobilization from BM, leading to reparative neovascularization and tissue repair. In pathophysiological states such as aging, atherosclerosis, heart failure, hypertension, and diabetes, excess amounts of ROS create an inflammatory and oxidative microenvironment, which induces cell damage and apoptosis of stem and progenitor cells. Understanding the molecular mechanisms of how ROS regulate the functions of stem and progenitor cells and their niche in physiological and pathological conditions will lead to the development of novel therapeutic strategies.     

Exposure to ambient particles accelerates monocyte release from bone marrow in atherosclerotic rabbits

Exposure to air pollution [particulate matter, particles <10 microm (PM(10))] causes a systemic inflammatory response that includes stimulation of the bone marrow (BM) and progression of atherosclerosis. Monocytes are known to play a key role in atherogenesis by migration into subendothelial lesions where they appear as foam cells. The present study was designed to quantify the BM monocyte response in Watanabe heritable hyperlipidemic (WHHL) rabbits after PM(10) exposure. WHHL rabbits were given twice weekly intrapharyngeal instillations of 5 mg of PM(10) for 4 wk to a total of 40 mg and compared with control WHHL or New Zealand White (NZW) rabbits. The thymidine analog 5'-bromo-2'-deoxyuridine was used to label dividing cells in the BM and a monoclonal antibody to identify monocytes in peripheral blood. The transit time of monocytes through the BM was faster in WHHL than in NZW rabbits (30.4 +/- 1.9 h vs. 35.2 +/- 0.9 h, WHHL vs. NZW; P < 0.05). PM(10) instillation exposure increased circulating band cell counts, caused rapid release of monocytes from the BM, and further shortened their transit time through the BM to 23.2 +/- 1.6 h (P < 0.05). The percentage of alveolar macrophages containing particles in the lung correlated with the BM transit time of monocytes (r(2) = 0.45, P <0.05). We conclude that atherosclerosis increases the release of monocytes from the BM, and PM(10) exposure accelerates this process in relation to the amount of particles phagocytosed by alveolar macrophages.     

Endothelial progenitor cells for regeneration

Endothelial progenitor cells (EPCs) have been recently isolated from peripheral blood and bone marrow (BM), and shown to be incorporated into sites of physiological and pathological neovascularization in vivo. In contrast to differentiated endothelial cells (ECs), transplantation of EPCs successfully enhanced vascular development by in situ differentiation and proliferation within ischemic organs. Based on such a novel concept of closed up function on EPCs in postnatal neovascularization, the beneficial property of EPC is attractive for cell therapy as well as cell-mediated gene therapy applications targeting regeneration of ischemic tissue.     

Bone marrow derived cells for brain repair: recent findings and current controversies

Adult stem cells were once thought to produce only the cell lineages characteristic of the tissues in which they reside. Recent studies suggest that cells derived from one adult tissue can be reprogrammed to change into cellular phenotypes not normally found in that tissue. Bone marrow (BM) derived cells have been demonstrated to differentiate into multiple lineages, including glial cells and neurons, both in vivo and in vitro. This unexpected plasticity of BM cells occurs not only under experimental conditions, but also in humans following BM transplantation. As a result, BM transplantation has emerged as a novel approach to enhance neural regeneration and restore injured brain tissue. Several research teams have reported that transplanted BM cells can differentiate into neural derivatives; indeed, some of these cells were capable of integration into the host brain, where they promoted functional recovery after brain injury. Other researchers conducting similar studies were unable to find any evidence of neural differentiation, concluding that differentiation 'from marrow to brain' is not a common phenomenon. More recently, two papers in Nature also cast doubt on the plasticity of adult stem cells, suggesting that the acquisition of different identities by grafted BM cells may merely reflect their fusion with host cells. Reasons for the wide discrepancies among findings in current BM stem cell research are unclear, making it difficult to understand the mechanisms by which transplanted marrow stem cells provide therapeutic benefit. Here, we summarize recent findings on this subject, and address some of the major controversies that have marked the evolution of adult stem cell research.     

Endothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance

Bone marrow and peripheral blood of adults contain a special sub-type of progenitor cells which are able to differentiate into mature endothelial cells, thus contributing to re-endothelialization and neo-vascularization. These angiogenic cells have properties of embryonal angioblasts and were termed endothelial progenitor cells (EPCs). In general, three surface markers (CD133, CD34 and the vascular endothelial growth factor receptor-2) characterize the early functional angioblast, located predominantly in the bone marrow. Later, when migrating to the systemic circulation EPCs gradually lose their progenitor properties and start to express endothelial marker like VE-cadherin, endothelial nitric oxide synthase and von Willebrand factor. The number of circulating EPCs in healthy subjects is rather low and a variety of conditions or factors may further influence this number. In the context of possible therapeutic application of EPCs recent clinical studies employing these cells for neo-vascularization of ischemic organs have just been published. However, the specificity of the observed positive clinical effects, the mechanisms regulating the differentiation of EPCs and their homing to sites of injured tissue remain partially unknown at present.     

Therapeutic effects of human STRO-3-selected mesenchymal precursor cells and their soluble factors in experimental myocardial ischemia

Stromal precursor antigen (STRO)-3 has previously been shown to identify a subset of adult human bone marrow (BM)-derived mesenchymal lineage precursors, which may have cardioprotective potential. We sought to characterize STRO-3-immunoselected and culture-expanded mesenchymal precursor cells (MPCs) with respect to their biology and therapeutic potential in myocardial ischemia. Immunoselection of STRO-3(+) MPCs enriched for fibroblastic colony forming units from unfractionated BM mononuclear cells (MNCs). Compared to mesenchymal stem cells conventionally isolated by plastic adherence, MPCs demonstrated increased proliferative capacity during culture expansion, expressed higher levels of early 'stem cell' markers and various pro-angiogenic and cardioprotective cytokines, and exhibited greater trilineage developmental efficiency. Intramyocardial injection of MPCs into a rat model of myocardial infarction (MI) promoted left ventricular recovery and inhibited left ventricular dilatation. These beneficial effects were associated with cardioprotective and pro-angiogenic effects at the tissue level, despite poor engraftment of cells. Treatment of MI rats with MPC-conditioned medium (CM) preserved left ventricular function and dimensions, reduced myocyte apoptosis and fibrosis, and augmented neovascularization, involving both resident vascular cells and circulating endothelial progenitor cells (EPCs). Profiling of CM revealed various cardioprotective and pro-angiogenic factors, which had biological activity in cultures of myocytes, tissue-resident vascular cells and EPCs. Prospective immunoselection of STRO-3(+) MPCs from BM MNCs conferred advantage in maintaining a population of immature MPCs during ex vivo expansion. Transplantation of culture-expanded MPCs into the post-MI heart resulted in therapeutic benefit, attributable at least in part to paracrine mechanisms of action. Thus, MPCs represent a promising therapy for myocardial ischemia.     

Oxidative stress-induced DNA damage by particulate air pollution

Exposure to ambient air particulate matter (PM) is associated with pulmonary and cardiovascular diseases and cancer. The mechanisms of PM-induced health effects are believed to involve inflammation and oxidative stress. The oxidative stress mediated by PM may arise from direct generation of reactive oxygen species from the surface of particles, soluble compounds such as transition metals or organic compounds, altered function of mitochondria or NADPH-oxidase, and activation of inflammatory cells capable of generating ROS and reactive nitrogen species. Resulting oxidative DNA damage may be implicated in cancer risk and may serve as marker for oxidative stress relevant for other ailments caused by particulate air pollution. There is overwhelming evidence from animal experimental models, cell culture experiments, and cell free systems that exposure to diesel exhaust and diesel exhaust particles causes oxidative DNA damage. Similarly, various preparations of ambient air PM induce oxidative DNA damage in in vitro systems, whereas in vivo studies are scarce. Studies with various model/surrogate particle preparations, such as carbon black, suggest that the surface area is the most important determinant of effect for ultrafine particles (diameter less than 100 nm), whereas chemical composition may be more important for larger particles. The knowledge concerning mechanisms of action of PM has prompted the use of markers of oxidative stress and DNA damage for human biomonitoring in relation to ambient air. By means of personal monitoring and biomarkers a few studies have attempted to characterize individual exposure, explore mechanisms and identify significant sources to size fractions of ambient air PM with respect to relevant biological effects. In these studies guanine oxidation in DNA has been correlated with exposure to PM(2.5) and ultrafine particles outdoor and indoor. Oxidative stress-induced DNA damage appears to an important mechanism of action of urban particulate air pollution. Related biomarkers and personal monitoring may be useful tools for risk characterization.     

Advanced glycation endproducts alter functions and promote apoptosis in endothelial progenitor cells through receptor for advanced glycation endproducts mediate overpression of cell oxidant stress

Endothelial progenitor cells (EPCs) play an important role in preventing atherosclerosis. The factors that regulate the function of EPCs are not completely clear. Increased formation of advanced glycation endproducts (AGEs) is generally regarded as one of the main mechanisms responsible for vascular damage in patients with diabetes and atherosclerosis. AGEs lead to the generation of reactive oxygen species (ROS) and part of the regenerative capacity of EPCs seems to be due to their low baseline ROS levels and reduced sensitivity to ROS-induced cell apoptosis. Therefore, we tested the hypothesis that AGEs can alter functions and promote apoptosis in EPCs through overpress cell oxidant stress. EPCs, isolated from bone marrow, were cultured in the absence or presence of AGEs (50, 100, and 200 μg/ml). A modified Boyden’s chamber was used to assess the migration of EPCs and the number of recultured EPCs was counted to measure the adhesiveness function. MTT assay was used to determine the proliferation function. ROS were analyzed using the ROS assay kit. A spectrophotometer was used to assess superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity, and PCR was used to test mRNA expression of SOD and GSH-PX. SiRNA was used to block receptor for advanced glycation endproducts (RAGEs) expression. Apoptosis was evaluated by Annexin V immunostaining and TUNEL staining. Co-culturing with AGEs increases ROS production, decreases anti-oxidant defenses, overpresses oxidant stress, inhibits the proliferation, migration, and adhesion of EPCs, and induces EPCs apoptosis. In addition, these effects were attenuated during block RAGE protein expression by siRNA. AGEs may serve to impair EPCs functions through RAGE-mediate oxidant stress, and promote EPCs sensitivity toward oxidative-stress-mediated apoptosis, which indicates a new pathophysiological mechanism of disturbed vascular adaptation in atherosclerosis and suggests that lower levels of AGEs might improve the success of progenitor cell therapy.     

Identification of very small embryonic like (VSEL) stem cells in bone marrow

Bone marrow (BM) develops in mammals by the end of the second/beginning of the third trimester of gestation and becomes a major hematopoietic organ in postnatal life. The alpha-chemokine stromal derived factor-1 (SDF-1) to CXCR4 ([Formula: see text]-protein-coupled seven transmembrane-spanning chemokine receptor) axis plays a major role in BM colonization by stem cells. By the end of the second trimester of gestation, BM becomes colonized by hematopoietic stem cells (HSC), which are chemoattracted from the fetal liver in a CXCR4-SDF-1-dependent manner. Whereas CXCR4 is expressed on HSC, SDF-1 is secreted by BM stroma and osteoblasts that line BM cavities. Mounting evidence indicates that BM also contains rare CXCR4(+) pluripotent stem cells (PSC). Recently, our group has identified a population of CXCR4(+) very small embryonic like stem cells in murine BM and human cord blood. We hypothesize that these cells are deposited during development in BM as a mobile pool of circulating PSC that play a pivotal role in postnatal tissue turnover, both of non-hematopoietic and hematopoietic tissues.     

Moderate dose insulin promotes function of endothelial progenitor cells

EPCs (endothelial progenitor cells) regenerate the vascular endothelial cells and keep the integrity of the vascular endothelium and thus may retard the onset of atherosclerosis. Steady state levels of EPCs in the circulation were found to be correlated with cardiovascular event risks. Given the close relationship between insulin and the cardiovascular system, we tested the long-term effects of moderate-dose insulin treatment on bone marrow-derived EPCs. Rat bone marrow EPCs were exposed to various levels of insulin under normal (5 mmol/l) or high (40 mmol/l) glucose conditions for 7 days. Insulin at levels near the physiological range (0.1, 1 nmol/l) up-regulated EPCs proliferation, stimulated NO (nitric oxide) production and reduced EPC senescence and ROS (reactive oxygen species) generation under both normal- and high-glucose conditions. Glucose exerted deleterious effects on EPCs contrary to insulin. Western blot analysis suggested concomitant decrease of Akt phosphorylation and eNOS (endothelial nitric oxide synthase) expression by high-glucose treatment and increase with insulin administration. Thus, insulin promoted several activities of EPCs, which suggested a potential endothelial protective role of insulin. Akt/eNOS pathway may be involved in the modulation of EPCs function by glucose and insulin.     

Id1 restrains p21 expression to control endothelial progenitor cell formation

Loss of Id1 in the bone marrow (BM) severely impairs tumor angiogenesis resulting in significant inhibition of tumor growth. This phenotype has been associated with the absence of circulating endothelial progenitor cells (EPCs) in the peripheral blood of Id1 mutant mice. However, the manner in which Id1 loss in the BM controls EPC generation or mobilization is largely unknown. Using genetically modified mouse models we demonstrate here that the generation of EPCs in the BM depends on the ability of Id1 to restrain the expression of its target gene p21. Through a series of cellular and functional studies we show that the increased myeloid commitment of BM stem cells and the absence of EPCs in Id1 knockout mice are associated with elevated p21 expression. Genetic ablation of p21 rescues the EPC population in the Id1 null animals, re-establishing functional BM-derived angiogenesis and restoring normal tumor growth. These results demonstrate that the restraint of p21 expression by Id1 is one key element of its activity in facilitating the generation of EPCs in the BM and highlight the critical role these cells play in tumor angiogenesis.     

Functional damage of endothelial progenitor cells is attenuated by 14-3-3-n through inhibition of mitochondrial injury and oxidative stress

Endothelial progenitor cells (EPCs) are precursor cells of vascular endothelial cells, which are widely involved in the pathological process of cardiovascular diseases. EPCs apoptosis could accelerate the process of cardiovascular diseases. 14-3-3-η protein has been proved to be a potent antiapoptosis molecule. However, inhibition of EPCs apoptosis by 14-3-3-η and further specific mechanism have not been investigated. EPCs were isolated from human cord blood, and identified using VEGFR2 and CD34. 14-3-3-η overexpression model in vitro was established. Cell invasion, apoptosis, and proliferation were measured by transwell, flow cytometry, and Cell Counting Kit-8, respectively. Expression of 14-3-3-η, Bcl-2, and voltage-dependent anion channel 1 (VDAC1) were measured using quantitative real-time polymerase chain reaction and western blot analysis. Reactive oxygen species (ROS) intensity was measured using 2ʹ-7ʹ dichlorofluorescin diacetate probe. Mitochondrial membrane potential was detected using JC-1 dye. Overexpression of 14-3-3-η significantly promoted invasion and proliferation, but suppressed apoptosis of EPCs. Overexpression of 14-3-3-η remarkably inhibited ROS and promoted antioxidant enzyme levels in EPCs. 14-3-3-η might inhibit apoptosis of EPCs through attenuating mitochondrial injury. This study might provide a new target, 14-3-3-η, for the prevention and treatment of cardiovascular diseases through targeting EPCs.     

Unique responses of stem cell-derived vascular endothelial and mesenchymal cells to high levels of glucose

Diabetes leads to complications in selected organ systems, and vascular endothelial cell (EC) dysfunction and loss is the key initiating and perpetuating step in the development of these complications. Experimental and clinical studies have shown that hyperglycemia leads to EC dysfunction in diabetes. 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. Whether these vascular stem/progenitor cells succumb to the adverse effects of high glucose remains unknown. We sought to determine whether adult vascular stem/progenitor cells display cellular activation and dysfunction upon exposure to high levels of glucose as seen in diabetic complications. Mononuclear cell fraction was prepared from adult blood and bone marrow. EPCs and MPCs were derived, characterized, and exposed to either normal glucose (5 mmol/L) or high glucose levels (25 mmol/L). We then assayed for cell activity and molecular changes following both acute and chronic exposure to high glucose. Our results show that high levels of glucose do not alter the derivation of either EPCs or MPCs. The adult blood-derived EPCs were also resistant to the effects of glucose in terms of growth. Acute exposure to high glucose levels increased caspase-3 activity in EPCs (1.4x increase) and mature ECs (2.3x increase). Interestingly, MPCs showed a transient reduction in growth upon glucose challenge. Our results also show that glucose skews the differentiation of MPCs towards the adipocyte lineage while suppressing other mesenchymal lineages. In summary, our studies show that EPCs are resistant to the effects of high levels of glucose, even following chronic exposure. The findings further show that hyperglycemia may have detrimental effects on the MPCs, causing reduced growth and altering the differentiation potential.