Phloroglucinol Inhibits the in vitro Differentiation Potential of CD34 Positive Cells into Endothelial Progenitor Cells

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 cells: therapeutic target for cardiovascular diseases

Circulating endothelial progenitor cells (EPCs) derived from bone marrow were isolated for the first time in 1997 and characterized. Recent evidence has indicated that EPCs contribute to re-endothelialization of injured vessels as well as neovascularization of ischemic lesions and that a decrease in the number of EPCs is an independent predictor of morbidity and mortality of cardiovascular diseases. These finding suggest that EPCs play a major role in the pathogenesis of atherosclerosis and cardiovascular diseases. Interestingly, the number and function of EPCs are regulated by not only various kinds of angiogenic cytokines and cardiovascular risk factors per se but also some interventions, including lifestyle modification (aerobic exercise, body weight loss, and smoking cessation) and pharmacological therapy (e.g., renin-angiotensin system inhibitor, statin, and erythropoietin). It is thought that regulation of the number and function of EPCs directly influences the maintenance and development of atherosclerosis. Therefore, it is clinically important to estimate the degree of EPC bioactivity and to increase the EPC bioactivity by appropriate interventions. In this review, we focus on the relationship between EPCs and cardiovascular risk factors and the role of EPCs in cardiovascular diseases.     

Endothelial nitric oxide synthase inhibition does not alter endothelial progenitor cell colony forming capacity or migratory activity

Endothelial nitric oxide synthase (eNOS) activity has been shown to play a pivotal role in the mobilization of endothelial progenitor cells (EPCs) into the circulation from bone marrow. Indeed, in eNOS-deficient mice, exercise-induced EPC mobilization is severely diminished. We determined ex vivo whether circulating EPC colony-forming capacity and migratory activity are influenced by eNOS activity. Peripheral-blood mononuclear cells were isolated from 20 healthy adults and preplated for 2 days, and nonadherent cells were further cultured for 7 days in the presence and absence of N-nitro-L-arginine methyl ester (L-NAME, 300 microM, an eNOS antagonist) to determine EPC colony-forming units. Migratory activity of EPCs, cultured with and without L-NAME (300 microM) was determined by utilizing a modified Boyden chamber. The number of EPC colony-forming units was not significantly different when cultured in the absence or presence of L-NAME (21+/-5 vs 18+/-5). Moreover, eNOS inhibition did not alter EPC migratory activity; mean fluorescence was similar in samples cultured with (983+/-126 RFUs) and without (962+/-105 RFUs) L-NAME. These in vitro results suggest that, in contrast to EPC mobilization from the bone marrow, eNOS does not exert a modulatory influence on the functional capacity of circulating EPCs to either form colonies or migrate.     

Circulating endothelial progenitor cells as biomarkers for prediction of cardiovascular outcomes

Experimental studies suggest that bone marrow-derived endothelial progenitor cells (EPCs) play an important role in the maintenance of endothelial integrity and hemostasis. The number of circulating EPC has been shown to be inversely correlated with cardiovascular risk factors and vascular function and to predict cardiovascular events independent of both traditional and non-traditional risk factors. Thus, EPCs provide a clinical advantage over the use of other biomarkers as their measurement is directly associated with endothelial function, and available evidence suggests that they are consistently and significantly associated with a spectrum of cardiovascular complications, such as acute coronary syndromes and coronary artery disease. However, many issues in the field of EPC isolation and identification, particularly in regards to the effective and unequivocal molecular characterization of these cells still remain unresolved. In addition, simple EPC counts do not adequately describe cardiovascular disease risk. This limitation is attributable to variation in the definition of EPCs, the number of existing cardiovascular risk factors in different patients as well as a difference in the interaction between EPCs and other hematopoietic progenitor, inflammatory cells or platelets.     

The effects of HMG-CoA reductase inhibitor on vascular progenitor cells

Circulating bone marrow-derived vascular progenitor cells contribute to angiogenesis, atherosclerosis, and the response to vascular injury. These vascular progenitor cells consist of two cell groups, endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs). Although HMG-CoA reductase inhibitors (statins) have been reported to inhibit atherosclerosis partially by increased EPCs, the effects of statins on SMPCs are unclear. Therefore, we investigated the relationship between EPCs and SMPCs and whether pravastatin has atheroprotective effects on SMPCs. Peripheral mononuclear cells (MNCs) were isolated and cultured on fibronectin-coated dishes in SMPC medium. MNCs were stained with acetylated low density lipoprotein and lectin, or alpha-smooth muscle actin, and cell numbers were counted. mRNA expression and vascular endothelial growth factor (VEGF) protein synthesis of MNCs were evaluated. Pravastatin significantly increased the number of EPC and decreased the number of SMPC. mRNA expression of VEGF, endothelial nitric oxide synthase, VEGF receptor-2 (KDR), and Akt were up-regulated, and VEGF secretion was increased by pravastatin. The present study demonstrated that pravastatin has promotive effects on the differentiation from MNCs to EPC cells, while inhibitory effects to SMPC cells. Our findings suggest a previously unreported mechanism of the effect of statin therapy on vascular progenitor cells.     

Additive effects of endothelial progenitor cells combined with ACE inhibition and beta-blockade on left ventricular function following acute myocardial infarction.

Animal studies have demonstrated the efficacy of endothelial progenitor cells (EPCs) in preventing left ventricular (LV) remodelling following myocardial infarction (MI). Preliminary human studies are underway, yet no studies have demonstrated efficacy in combination with standard medical therapy, i.e. angiotensin-converting enzyme (ACE) inhibitors and beta-blockers. Nude rats underwent left anterior descending coronary artery ligation to induce MI. Animals were randomised to receive no treatment (MI, n = 5), quinapril 200 mg/L + metoprolol 2 g/L (ACE/BB, n = 5), two million EPCs intravenously (EPC, n = 5)or both (ACE/BB + EPC [n = 5]), then sacrificed after two weeks treatment. ACE/BB resulted in a 75% reduction in fibrosis in the region remote from the MI (p < 0.05), but EPC therapy had little effect here. Conversely, EPC therapy induced neovascularisation at the peri-infarct rim, thereby preventing peri-infarct apoptosis by 81% (p < 0.05). Acting via different but complementary mechanisms, the combination of ACE/BB + EPCs resulted in a greater overall improvement in LV function on echocardiography than either therapy alone. Clinical trials using stem cell therapy in conjunction with standard medical treatment are warranted.     

The effects of growth hormone and insulin-like growth factor-1 on the aging cardiovascular system and its progenitor cells

Aging is a major risk factor for the development of cardiovascular disease. Aging is also associated with a decline in the growth hormone (GH) and insulin-like growth factor-1 (IGF-1) axis. This axis impacts endothelial and vascular smooth muscle cell biology, as well as cardiac function. The number of endothelial progenitor cells (EPCs) also decreases with age and is emerging as a surrogate measurement of vascular senescence. Studies suggest that EPCs impact vascular health by modulating vascular repair and function. Current evidence demonstrates that EPC number and function is restored with a GH-mediated increase in serum IGF-1. Modulation of the GH and IGF-1 system may therefore provide a useful therapy in the prevention of age-associated changes in the cardiovascular system and in future regenerative cell-based therapies.     

The pleiotropic effects of ARB in vascular endothelial progenitor cells

Angiotensin II regulates blood pressure and contributes to endothelial dysfunction and the progression of atherosclerosis. Bone marrow-derived endothelial progenitor cells (EPCs) in peripheral blood contribute to postnatal vessel repair and neovascularization. Impaired EPC function in patients with hypertension and diabetes inhibits the endogenous repair of vascular lesions and leads to the progression of atherosclerosis. The number of EPCs in peripheral blood is inversely correlated with mortality and the occurrence of cardiovascular events. Angiotensin II-mediated signaling is implicated in oxidative stress, inflammation and insulin resistance, factors that cause EPC dysfunction. Blockade of the angiotensin II type 1 receptor may therefore present a new therapeutic target for enhancing EPC function.     

Dysfunction of endothelial progenitor cells under diabetic conditions and its underlying mechanisms

Cardiovascular complications have been major concerns in the treatment of diabetes, and up to 80% of all deaths in diabetic patients are linked to cardiovascular problems. Impaired angiogenesis is one of the most serious symptoms associated with diabetes, resulting in delayed wound healing and lower limb amputation. Endothelial progenitor cells (EPCs), a subpopulation of adult stem cells, are recruited from bone marrow to the injured vessel to promote endothelial regeneration and neovascularization, playing an important role in angiogenesis. Interestingly, several clinical studies have showed that the number of recruited EPCs is reduced and their function is decreased under diabetic conditions, implying that diabetic EPC dysfunction may contribute to defective angiogenesis and resultant cardiovascular complications in diabetes. To recover the functional abilities of diabetic EPCs and to address possible application of EPC cell therapy to diabetic patients, some studies provided explanations for diabetic EPC dysfunction including increased oxidative stress, involvement of the inflammatory response, alteration in the nitric oxide pathway and reduced signals for EPC recruitment. This review discusses clinical evidence of impairment of EPC functions under diabetic conditions and the suggested mechanisms for diabetic EPC dysfunction.     

The acute exposure effects of inhaled nickel nanoparticles on murine endothelial progenitor cells

Abstract

Introduction: The discovery of endothelial progenitor cells (EPCs) may help to explain observed cardiovascular effects associated with inhaled nickel nanoparticle exposures, such as increases in vascular inflammation, generation of reactive oxygen species, altered vasomotor tone and potentiated atherosclerosis in murine species.

Methods: Following an acute whole body inhalation exposure to 500?µg/m³ of nickel nanoparticles for 5?h, bone marrow EPCs from C57BL/6 mice were isolated. EPCs were harvested for their RNA or used in a variety of assays including chemotaxis, tube formation and proliferation. Gene expression was assessed for important receptors involved in EPC mobilization and homing using RT-PCR methods. EPCs, circulating endothelial progenitor cells (CEPCs), circulating endothelial cells (CECs) and endothelial microparticles (EMPs) were quantified on a BD FACSCalibur to examine endothelial damage and repair associated with the exposure.

Results and conclusions: Acute exposure to inhaled nickel nanoparticles significantly increased both bone marrow EPCs as well as their levels in circulation (CEPCs). CECs were significantly elevated indicating that endothelial damage occurred due to the exposure. There was no significant difference in EMPs between the two groups. Tube formation and chemotaxis, but not proliferation, of bone marrow EPCs was impaired in the nickel nanoparticle exposed group. These results coincided with a decrease in the mRNA of receptors involved in EPC mobilization and homing. These data provide new insight into how an acute nickel nanoparticle exposure to half of the current Occupational Safety & Health Administration (OSHA) permissible exposure limit may adversely affect EPCs and exacerbate cardiovascular disease states.     

Effects of puerarin on number and activity of endothelial progenitor cells from peripheral blood

AIM: To investigate whether puerarin can augment endothelial progenitor cells (EPCs) numbers, promote EPC proliferative, migratory, adhesive, and in vitro vasculogenesis capacity. METHODS: EPCs were characterized as adherent cells by double staining of DiLDL-uptake and lectin binding under a laser scanning confocal microscope. Expression of KDR, VEGFR-2, and AC 133 was detected by flow cytometry. EPCs proliferation, migration and in vitro vasculogenesis were determined with MTT assay, modified Boyden chamber assay, and in vitro vasculogenesis kit, respectively. EPCs adhesive assay was performed by replating those on fibronectin-coated dishes, then adherent cells were counted. RESULTS: Incubation of isolated human MNCs with puerarin 0.1-3 mmol/L increased the number of EPCs, EPC proliferative, migratory, adhesive, and in vitro vasculogenesis capacity in a concentration and time-dependent manner, which reached maximum at 3 mmol/L, 24 h (approximately 1-fold increase, P<0.01). CONCLUSION: Puerarin enhanced     

同型半胱氨酸对内皮祖细胞增殖 黏附和凋亡的影响

目的观察不同浓度同型半胱氨酸(Hcy)对内皮祖细胞(EPCs)功能的影响。方法密度梯度离心法获取单个核细胞,培养7d后,收集贴壁细胞。在多波长激光共聚焦显微镜下,呈异硫氰酸荧光素标记的荆豆凝集素(FITC-UEA-I)和DiI标记的乙酰化低密度脂蛋白(DiI-acLDL)双染色阳性者为正在分化的EPCs,流式细胞仪检测其表面标志进一步鉴定EPCs。不同浓度同型半胱氨酸组(10、50、100、200μmol/L)干预24h,分别测定EPCs的增殖、黏附和凋亡。结果与对照组比较,Hcy组呈剂量依赖性地损害EPCs的增殖、黏附,促进其凋亡。200μmol/L的Hcy组对EPCs影响最为显著(P<0.01)。结论Hcy损害EPCs的功能。     

AMD3100 mobilizes endothelial progenitor cells in mice, but inhibits its biological functions by blocking an autocrine/paracrine regulatory loop of stromal cell derived factor-1 in vitro

Endothelial progenitor cells (EPCs), a CXCR4-bearing cell line, are thought to positively influence reendothelialization, vascular repair, and angiogenesis. AMD3100, a highly selective antagonist of stromal cell derived factor (SDF)-1 that binds to its receptor, CXCR4, has been shown to induce rapid mobilization of hematopoietic stem cells in mice, dogs, and humans. Results of this study indicate that AMD3100 injection can induce a rapid and potent mobilization of EPCs in mice compared with saline injection. The study demonstrates that murine spleen-derived EPCs can produce high levels of SDF-1 in vitro. Blocking this endogenous SDF-1 with AMD3100 decreases the number of EPCs; inhibits the proliferation, migration, and adhesion of EPCs; and induces EPC apoptosis. In addition, AMD3100 inhibits ectogenous SDF-1alpha induced improvement of EPC functions. However, AMD3100 incubation does not change the functions of CXCR4-negative cell line HPDE6. In conclusion, AMD3100 can be regarded as a potent mobilizer of EPCs in mice. EPCs have an autocrine/paracrine SDF-1 loop, and breaking this loop with AMD3100 can inhibit the EPC functions in vitro.     

The effect of acute exposure to coarse particulate matter air pollution in a rural location on circulating endothelial progenitor cells: results from a randomized controlled study

Abstract

Context: Fine particulate matter (PM) air pollution has been associated with alterations in circulating endothelial progenitor cell (EPC) levels, which may be one mechanism whereby exposures promote cardiovascular diseases. However, the impact of coarse PM on EPCs is unknown.

Objective: We aimed to determine the effect of acute exposure to coarse concentrated ambient particles (CAP) on circulating EPC levels.

Methods: Thirty-two adults (25.9?±?6.6 years) were exposed to coarse CAP (76.2?±?51.5?μg?m^?3) in a rural location and filtered air (FA) for 2 h in a randomized double-blind crossover study. Peripheral venous blood was collected 2 and 20 h post-exposures for circulating EPC (n?=?21), white blood cell (n?=?24) and vascular endothelial growth factor (VEGF) (n?=?16–19) levels. The changes between exposures were compared by matched Wilcoxon signed-rank tests.

Results: Circulating EPC levels were elevated 2 [108.29 (6.24–249.71) EPC?mL^?1; median (25th–75th percentiles), p?=?0.052] and 20 h [106.86 (52.91–278.35) EPC?mL^?1, p?=?0.008] post-CAP exposure compared to the same time points following FA [38.47 (0.00–84.83) and 50.16 (0.00–104.79) EPC?mL^?1]. VEGF and white blood cell (WBC) levels did not differ between exposures.

Conclusions: Brief inhalation of coarse PM from a rural location elicited an increase in EPCs that persisted for at least 20 h. The underlying mechanism responsible may reflect a systemic reaction to an acute “endothelial injury” and/or a circulating EPC response to sympathetic nervous system activation.     

Effects of resveratrol on endothelial progenitor cells and their contributions to reendothelialization in intima-injured rats

The aim of this study was to investigate the effects of resveratrol on endothelial progenitor cell (EPC) activities in vitro and on the mobilization of circulating EPCs, and reendothelialization in balloon-injured aorta of rats. After being isolated, cultured, and characterized, human EPCs were stimulated with resveratrol. We found that a low concentration of resveratrol (1 microM) led to significant enhanced activities of proliferation, migration, and adhesion, as well as promoting endothelial nitric acid synthetase (eNOS) expression in EPCs, whereas a high concentration (60 microM) inhibited the aforementioned functions and eNOS expression. In a rat model of injured aorta, a low dosage of resveratrol (10 mg/kg) increased the amount of EPCs in rat circulation as compared with placebo, whereas the result of a high dosage (50 mg/kg) did not reach statistical difference. In addition, 10 mg/kg of resveratrol both accelerated reendothelialization and inhibited neointimal formation; however, 50 mg/kg only reduced neointimal formation, which was not as effective as the previous one. eNOS expression in injured arteries was potently enhanced in the 10 mg/kg group, but not in the 50 mg/kg group.These findings suggest that a low dosage of resveratrol could markedly raise the proliferative, migrative, and adhesive activities of EPCs and upgrade eNOS expression in vitro as well as increase EPC mobilization, enhance eNOS expression, and accelerate the repair of injured artery; however, a high dosage cannot.     

Effects of metoprolol,methyldopa, and nifedipine on endothelial progenitor cells in patients with gestational hypertension and preeclampsia

Endothelial progenitor cells (EPCs) are critical for vascular regeneration and function, but are reduced in hypertensive disorders of pregnancy. We aimed to determine the possible effects of antihypertensive drugs, such as metoprolol, methyldopa, and nifedipine, on EPC number and functions in patients with gestational hypertension and preeclampsia. We collected blood samples from 30 normal pregnant women, 67 patients with gestational hypertension and 48 patients with preeclampsia. The patients received no drug or an antihypertensive drug, such as metoprolol, methyldopa, or nifedipine, between 20 and 24 weeks of gestation. The number of EPCs and circulating endothelial cells (CECs) in the blood was measured by flow cytometry. Moreover, colony formation and migration assays were performed on the isolated EPCs. Both the systolic and diastolic blood pressure (BP) increased, while the percentage of flow‐mediated vasodilatation (FMD) decreased in patients with gestational hypertension and preeclampsia, compared to the healthy controls at 20 weeks of gestation. CEC number increased in the patients, whereas EPC counts decreased. Furthermore, EPC colony formation and migration abilities were also impaired in the patients. However, administration of metoprolol, methyldopa, or nifedipine effectively restored the systolic and diastolic BP, FMD%, EPCs, and CEC numbers, as well as EPC migration capacity. Endothelial progenitor cells colony formation ability selectively improved with methyldopa and nifedipine. In patients receiving no drugs, most of these indexes worsened within 4 weeks (study duration). This study revealed a new pharmacological action of these antihypertensive drugs against gestational hypertension and preeclampsia, thus supporting their clinical use.     

Stimulation of endothelial progenitor cells: a new putative effect of several cardiovascular drugs

The role of vascular endothelium in cardiovascular disorders is well recognized. Mature endothelial cells contribute to the repair of endothelial injury, but they only have a limited capacity to do so. This has led to growing interest and further investigation into circulating endothelial progenitor cells (EPCs) and their role in vascular healing, repair, and postnatal neovascularization. The current perception of vascular health is that of a balance between ongoing injury and resultant vascular repair, mediated at least in part by circulating EPCs. Circulating EPCs play an important role in accelerating endothelialization at areas of vascular damage, and EPC enumeration is a viable strategy for assessing reparative capacity. Recent studies have shown that EPCs are affected both in number and function by several cardiovascular risk factors as well as various cardiovascular disease states, such as hypertension, hypercholesterolemia, and coronary artery disease. The present review summarizes the most relevant studies on the effects of cardiovascular drugs on vascular function and EPCs, focusing on their mechanisms of action.     

Short-term statin discontinuation increases endothelial progenitor cells without inflammatory rebound in type 2 diabetic patients

Type 2 diabetes (T2D) is characterized by impaired vascular regeneration owing to reduced endothelial progenitor cells (EPCs). While statins are known to increase EPCs, the effects of statin withdrawal on EPCs are unknown. Herein, we evaluated the effects of statin discontinuation on EPCs, inflammation and in vivo angiogenesis. Thirty-four T2D patients were randomized to 5-day discontinuation or continuation of statin treatment. At baseline and at day 5, we determined lipid profile, EPC levels, monocyte–macrophage polarization, and concentrations of hsCRP, VEGF, SDF-1α, and G-CSF. Angiogenesis by human circulating cells was assessed in vivo. At day 5, patients who stopped statins showed raised total and LDL cholesterol and EPCs compared to baseline, while no changes were observed in patients who continued statins. No changes were observed in hsCRP, VEGF, SDF-1α, G-CSF, M1 and M2 macrophages and classical, intermediate and nonclassical monocytes in both groups. In vivo angiogenesis by circulating cells was increased in patients who stopped statin treatment. In vitro, cholesterol supplementation stimulated mobilizing signals in human bone marrow mesenchymal stem cells. In conclusion, a brief statin withdrawal increases circulating EPCs and functional proangiogenic cells in T2D. These findings identify statin-sensitive pathways as reverse target mechanisms to stimulate vascular repair in diabetes.     

糖尿病患者血管内皮祖细胞研究进展

血管内皮祖细胞(EPC)来源于骨髓,是具有修复内皮和新生血管功能的干细胞。糖尿病患者外周血EPC数量和功能均出现下降,EPC已成为糖尿病及其并发症治疗的一个新靶点。本文综述EPC在糖尿病病理生理中的作用和药物干预机理的研究进展。     

Dronedarone hydrochloride enhances the bioactivity of endothelial progenitor cells via regulation of the AKT signaling pathway

Cardiovascular disease (CVD) and its complications are the leading cause of morbidity and mortality in the world. Because of the side effects and incomplete recovery from current therapy, stem cell therapy emerges as a potential therapy for CVD treatment, and endothelial progenitor cell (EPC) is one of the key stem cells used for therapeutic applications. The effect of this therapy required the expansion of EPC function. To enhance the EPC activation, proliferation, and angiogenesis using dronedarone hydrochloride (DH) is the purpose of this study. DH received approval for atrial fibrillation treatment and its cardiovascular protective effects were already reported. In this study, DH significantly increased EPC proliferation, tube formation, migration, and maintained EPCs surface marker expression. In addition, DH treatment up-regulated the phosphorylation of AKT and reduced the reactive oxygen species production. In summary, the cell priming by DH considerably improved the functional activity of EPCs, and the use of which might be a novel strategy for CVD treatment.