Cyclosporine increases ischemia-induced endothelial progenitor cell mobilization through manipulation of the CD26 system

Cyclosporin A (CsA) improves the success rate of transplantation. The CD26/dipeptidylpeptidase IV (DPP IV) system plays a critical role in mobilizing endothelial progenitor cells (EPCs) from bone marrow. This study investigated whether CsA manipulates CD26/DPP IV activity and increases EPC mobilization. C57BL/6 mice were divided into control and CsA-treated groups. Before and after hindlimb ischemia was induced, circulating EPC number and serum levels of different cytokines were measured. Compared with the controls, CsA treatment significantly increased the blood levels of stroma-derived factor-1alpha and stem cell factor after ischemic stress (P < 0.001). The CsA group displayed a significant increase in the number of circulating EPCs (sca-1+KDR+ and c-kit+CD31+ EPCs, both P < 0.05). In vivo, CsA caused a significant increase in the numbers of EPCs incorporated into the Matrigel and ischemic limbs (P < 0.05). In the peripheral blood, CsA significantly decreased CD26+ cell numbers and attenuated the plasma CD26/DPP IV activity (P < 0.001). Furthermore, short-term CsA treatment significantly improved the perfusion of ischemic limbs and decreased the spontaneous digital amputation rate. In summary, CsA manipulates the mobilization of EPCs into the circulation via the CD26/DPP IV system. Short-term CsA treatment has beneficial effects on angiogenesis of ischemic tissues.     

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.     

Circulating endothelial progenitor cells in kidney transplant patients

Background

Kidney transplantation (RTx) leads to amelioration of endothelial function in patients with advanced renal failure. Endothelial progenitor cells (EPCs) may play a key role in this repair process. The aim of this study was to determine the impact of RTx and immunosuppressive therapy on the number of circulating EPCs.

Methods

We analyzed 52 RTx patients (58±13 years; 33 males, mean ± SD) and 16 age- and gender-matched subjects with normal kidney function (57±17; 10 males). RTx patients received a calcineurin inhibitor (CNI)-based (65%) or a CNI-free therapy (35%) and steroids. EPC number was determined by double positive staining for CD133/VEGFR2 and CD34/VEGFR2 by flow cytometry. Stromal cell-derived factor 1 alpha (SDF-1) levels were assessed by ELISA. Experimentally, to dissociate the impact of RTx from the impact of immunosuppressants, we used the 5/6 nephrectomy model. The animals were treated with a CNI-based or a CNI-free therapy, and EPCs (Sca+cKit+) and CD26+ cells were determined by flow cytometry.

Results

Compared to controls, circulating number of CD34+/VEGFR2+ and CD133+/VEGFR2+ EPCs increased in RTx patients. There were no correlations between EPC levels and statin, erythropoietin or use of renin angiotensin system blockers in our study. Indeed, multivariate analysis showed that SDF-1 – a cytokine responsible for EPC mobilization – is independently associated with the EPC number. 5/6 rats presented decreased EPC counts in comparison to control animals. Immunosuppressive therapy was able to restore normal EPC values in 5/6 rats. These effects on EPC number were associated with reduced number of CD26+ cells, which might be related to consequent accumulation of SDF-1.

Conclusions

We conclude that kidney transplantation and its associated use of immunosuppressive drugs increases the number of circulating EPCs via the manipulation of the CD26/SDF-1 axis. Increased EPC count may be associated to endothelial repair and function in these patients.     

CXCR2-Dependent Endothelial Progenitor Cell Mobilization in Pancreatic Cancer Growth

Neovascularization is essential for tumor growth. We have previously reported that the chemokine receptor CXCR2 is an important regulator in tumor angiogenesis. Here we report that the mobilization of bone marrow (BM)-derived endothelial progenitor cells (EPCs) is impaired in CXCR2 knockout mice harboring pancreatic cancers. The circulating levels of EPCs (positive for CD34, CD117, CD133, or CD146) are decreased in the bone marrow and/or blood of tumor-bearing CXCR2 knockout mice. CXCR2 gene knockout reduced BM-derived EPC proliferation, differentiation, and vasculogenesis in vitro. EPCs double positive for CD34 and CD133 increased tumor angiogenesis and pancreatic cancer growth in vivo. In addition, CD133(+) and CD146(+) EPCs in human pancreatic cancer are increased compared with normal pancreas tissue. These findings indicate a role of BM-derived EPC in pancreatic cancer growth and provide a cellular mechanism for CXCR2 mediated tumor neovascularization.     

Haematopoietic stem cells and endothelial progenitor cells in healthy men: effect of aging and training

The number of hematopoietic stem cells (HSC) and endothelial progenitor cells (EPC) is thought to be a marker for neovascularization and vascular repair. Because physical inactivity and aging are risk factors for cardiovascular diseases, these factors may influence the numbers of HSCs and EPCs. Therefore, we examined baseline and exercise-induced levels of HSCs and EPCs in sedentary and trained young and older men. To study the role of aging in eight sedentary young (19-28 years) and eight sedentary older men (67-76 years), baseline and acute exercise-induced numbers of HSCs (CD34+-cells) and EPCs (CD34+/VEGFR-2+-cells) were quantified by fluorescence-activated cell sorter (FACS) analysis. To examine the effect of chronic training, eight age-matched trained young men (18-28 years) were compared with sedentary young men, whereas older men performed an 8-week endurance training. Older men showed significantly lower baseline and exercise-induced levels of HSCs/EPCs than the young men (P < 0.05). In young and older men, acute exercise significantly increased HSCs (P < 0.01), but not EPCs. The absolute increase in numbers of HSCs was attenuated in older men (P = 0.03). Apart from the lower baseline numbers of EPCs after chronic training in older men, training status did not alter baseline or exercise-induced levels of HSCs/EPCs in young and older men. We concluded that advancing age results in lower circulating numbers of HSCs and EPCs and attenuates the acute exercise-induced increase in HSCs. Interestingly, in young as well as in older men chronic endurance training does not affect baseline and exercise-induced numbers of HSCs and EPCs.     

Decursin inhibits vasculogenesis in early tumor progression by suppression of endothelial progenitor cell differentiation and function

Endothelial progenitor cells (EPCs) contribute to the tumor vasculature during tumor progression. Decursin isolated from the herb Angelica gigas is known to possess potent anti‐inflammatory activities. Recently, we reported that decursin is a novel candidate for an angiogenesis inhibitor [Jung et al., 2009 ]. In this study, we investigated whether decursin regulates EPC differentiation and function to inhibit tumor vasculogenesis. We isolated AC133+ cells from human cord blood and decursin significantly decreased the number of EPC colony forming units of human cord blood‐derived AC133+ cells that produce functional EPC progenies. Decursin dose‐dependently decreased the cell number of EPC committing cells as demonstrated by EPC expansion studies. Decursin inhibited EPC differentiation from progenitor cells into spindle‐shaped EPC colonies. Additionally, decursin inhibited proliferation and migration of early EPCs isolated from mouse bone marrow. Furthermore, decursin suppressed expression of angiopoietin‐2, angiopoietin receptor Tie‐2, Flk‐1 (vascular endothelial growth factor receptor‐2), and endothelial nitric oxide synthase in mouse BM derived EPCs in a dose‐dependent manner. Decursin suppressed tube formation ability of EPCs in collaboration with HUVEC. Decursin (4 mg/kg) inhibited tumor‐induced mobilization of circulating EPCs (CD34 + /VEGFR‐2+ cells) from bone marrow and early incorporation of Dil‐Ac‐LDL‐labeled or green fluorescent protein (GFP)+ EPCs into neovessels of xenograft Lewis lung carcinoma tumors in wild‐type‐ or bone‐marrow‐transplanted mice. Accordingly, decursin attenuated EPC‐derived endothelial cells in neovessels of Lewis lung carcinoma tumor masses grown in mice. Together, decursin likely affects EPC differentiation and function, thereby inhibiting tumor vasculogenesis in early tumorigenesis. J. Cell. Biochem. 113: 1478–1487, 2012. © 2012 Wiley Periodicals, Inc.     

Antioxidants increase number of progenitor endothelial cells through multiple gene expression pathways

To date, there is no report on the effect of antioxidants on endothelial progenitor cells (EPCs). This study shows that in vitro incubation of EPCs with vitamin C and E reverted the already well documented lowering effect of TNF-α on EPC number and increased p-p38 expression levels. In order to document major changes of gene expression levels and gain insight into signalling pathways, microarray analysis was performed and a significant variation of the expression of 5389 genes in EPCs following antioxidant treatment was detected. Also in vivo evidence is provided about the positive effect of antioxidant vitamins on EPCs, since vitamin C and E supplementation potentiated the physical training-induced increase of EPC number and VEGF levels. Together, these data indicate that antioxidant treatment ameliorates EPC number and causes major changes of gene expression within these cells in vitro. Furthermore, concomitant antioxidant supplementation and physical training in vivo raised the levels of circulating EPCs and serum VEGF more than physical training alone.     

Successful in vitro expansion and differentiation of cord blood derived CD34+ cells into early endothelial progenitor cells reveals highly differential gene expression

Endothelial progenitor cells (EPCs) can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU) assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP), PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15) or pro-angiogenic (galectin-3) properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP) was the most up-regulated gene.     

Comparison between total endothelial progenitor cell isolation versus enriched Cd133+ culture

Endothelial progenitor cells (EPCs) play a role in endogenous neovascularization of ischaemic tissues. Isolation and characterization of EPCs from circulating mononuclear cells are important for developing targeted cellular therapies and reproducibility of data are the major scientific goals. Here we compared two currently employed isolation methods, i.e. from total peripheral blood mononuclear cells (PBMCs) and from enriched CD133(+) cells, by defining the cell morphology and functional activities. We show that EPCs from cultured PBMCs resulted in an adherent population of 23% +/- 4% merged cells positive for Dil-Ac-LDL and lectin, whereas the percentage of double positive cells in cultured CD133(+) enriched cells was 50% +/- 7% (P < 0.01). These data were obtained through a novel and a more complete method of analysis of cell calculations (specifically by dividing each microscope field into 120 subfields). When stimulated with tumour necrosis factor alpha (TNF)-alpha and glucose, cell number was reduced in EPCs from total PBMCs and, more consistently, in CD133(+) enriched cells. However, both cultured total PBMCs and CD133(+) enriched cells respond similarly to TNF-alpha or glucose-induced p38-phosphorylation. EPCs from both procedures show similar results in terms of phenotype and response to modulators of their functional activities. However, when the cell phenotype of CD133(+) enrichment-derived cells was compared with that of cells from the total PBMC, a significant increase in CD133(+) expression was observed (P < 0.01) This may have relevance during intervention studies using cultured EPCs.     

Hyperoxia reduces bone marrow, circulating, and lung endothelial progenitor cells in the developing lung: implications for the pathogenesis of bronchopulmonary dysplasia

Hyperoxia disrupts vascular and alveolar growth of the developing lung and contributes to the development of bronchopulmonary dysplasia (BPD). Endothelial progenitor cells (EPC) have been implicated in repair of the vasculature, but their role in lung vascular development is unknown. Since disruption of vascular growth impairs lung structure, we hypothesized that neonatal hyperoxia impairs EPC mobilization and homing to the lung, contributing to abnormalities in lung structure. Neonatal mice (1-day-old) were exposed to 80% O(2) at Denver's altitude (= 65% at sea level) or room air for 10 days. Adult mice were also exposed for comparison. Blood, lung, and bone marrow were harvested after hyperoxia. Hyperoxia decreased pulmonary vascular density by 72% in neonatal but not adult mice. In contrast to the adult, hyperoxia simplified distal lung structure neonatal mice. Moderate hyperoxia reduced EPCs (CD45-/Sca-1+/CD133+/VEGFR-2+) in the blood (55%; P < 0.03), bone marrow (48%; P < 0.01), and lungs (66%; P < 0.01) of neonatal mice. EPCs increased in bone marrow (2.5-fold; P < 0.01) and lungs (2-fold; P < 0.03) of hyperoxia-exposed adult mice. VEGF, nitric oxide (NO), and erythropoietin (Epo) contribute to mobilization and homing of EPCs. Lung VEGF, VEGF receptor-2, endothelial NO synthase, and Epo receptor expression were reduced by hyperoxia in neonatal but not adult mice. We conclude that moderate hyperoxia decreases vessel density, impairs lung structure, and reduces EPCs in the circulation, bone marrow, and lung of neonatal mice but increases EPCs in adults. This developmental difference may contribute to the increased susceptibility of the developing lung to hyperoxia and may contribute to impaired lung vascular and alveolar growth in BPD.     

Diabetes alters subsets of endothelial progenitor cells that reside in blood, bone marrow, and spleen

Circulating endothelial progenitor cells (EPCs) derived from the bone marrow (BM) participate in maintaining endothelial integrity and vascular homeostasis. Reduced EPC number and function result in vascular complications in diabetes. EPCs are a population of cells existing in various differentiation stages, and their cell surface marker profiles change during the process of mobilization and maturation. Hence, a generally accepted marker combination and a standardized protocol for the quantification of EPCs remain to be established. To determine the EPC subsets that are affected by diabetes, we comprehensively analyzed 32 surface marker combinations of mouse peripheral blood (PB), BM, and spleen cells by multicolor flow cytometry. Ten subsets equivalent to previously reported mouse EPCs significantly declined in number in the PB of streptozotocin-induced diabetic mice, and this reduction was reversed by insulin treatment. The PI(-)Lin(-)c-Kit(-)Sca-1(+)Flk-1(-)CD34(-)CD31(+) EPC cluster, which can differentiate into mature endothelial cells in vitro, was the highest population in the PB, BM, and spleen and occurred 61 times more in the spleen than in the PB. The cell number significantly decreased in the BM as well as in the PB but paradoxically increased in the spleen under diabetic conditions. Insulin treatment reversed the decrease of EPC subsets in the BM and PB and reversed their increase in spleen. A similar tendency was observed in some of the major cell populations in db/db mice. To the best of our knowledge, we are the first to report spatial population changes in mouse EPCs by diabetes in the blood and in the BM across the spleen. Diminished circulating EPC supply by diabetes may be ascribed to impaired EPC production in the BM and to decreased EPC mobilization from the spleen, which may contribute to vascular dysfunction in diabetic conditions.     

Decreased circulating endothelial progenitor cell levels and function in patients with nonalcoholic fatty liver disease

Objectives

Nonalcoholic fatty liver disease (NAFLD) is associated with advanced atherosclerosis and a higher risk of cardiovascular disease. Increasing evidence suggests that injured endothelial monolayer is regenerated by circulating bone marrow derived-endothelial progenitor cells (EPCs), and levels of circulating EPCs reflect vascular repair capacity. However, the relation between NAFLD and EPC remains unclear. Here, we tested the hypothesis that patients with nonalcoholic fatty liver disease (NAFLD) might have decreased endothelial progenitor cell (EPC) levels and attenuated EPC function.

Methods and Results

A total of 312 consecutive patients undergoing elective coronary angiography because of suspected coronary artery disease were screened and received examinations of abdominal ultrasonography between July 2009 and November 2010. Finally, 34 patients with an ultrasonographic diagnosis of NAFLD, and 68 age- and sex-matched controls without NAFLD were enrolled. Flow cytometry with quantification of EPC markers (defined as CD34⁺, CD34⁺KDR⁺, and CD34⁺KDR⁺CD133⁺) in peripheral blood samples was used to assess circulating EPC numbers. The adhesive function, and migration, and tube formation capacities of EPCs were also determined in NAFLD patients and controls. Patients with NAFLD had a significantly higher incidence of metabolic syndrome, previous myocardial infarction, hyperuricemia, and higher waist circumference, body mass index, fasting glucose and triglyceride levels. In addition, patients with NAFLD had significantly decreased circulating EPC levels (all P<0.05), attenuated EPC functions, and enhanced systemic inflammation compared to controls. Multivariate logistic regression analysis showed that circulating EPC level (CD34⁺KDR⁺ [cells/10⁵ events]) was an independent reverse predictor of NAFLD (Odds ratio: 0.78; 95% confidence interval: 0.69–0.89, P<0.001).

Conclusions

NAFLD patients have decreased circulating EPC numbers and functions than those without NAFLD, which may be one of the mechanisms to explain atherosclerotic disease progression and enhanced cardiovascular risk in patients with NAFLD.     

GroEL1, a Heat Shock protein 60 of Chlamydia pneumoniae,Impairs Neovascularization by Decreasing Endothelial Progenitor Cell Function

The number and function of endothelial progenitor cells (EPCs) are sensitive to hyperglycemia, hypertension, and smoking in humans, which are also associated with the development of atherosclerosis. GroEL1 from Chlamydia pneumoniae has been found in atherosclerotic lesions and is related to atherosclerotic pathogenesis. However, the actual effects of GroEL1 on EPC function are unclear. In this study, we investigate the EPC function in GroEL1-administered hind limb-ischemic C57BL/B6 and C57BL/10ScNJ (a toll-like receptor 4 (TLR4) mutation) mice and human EPCs. In mice, laser Doppler imaging, flow cytometry, and immunohistochemistry were used to evaluate the degree of neo-vasculogenesis, circulating level of EPCs, and expression of CD34, vWF, and endothelial nitric oxide synthase (eNOS) in vessels. Blood flow in the ischemic limb was significantly impaired in C57BL/B6 but not C57BL/10ScNJ mice treated with GroEL1. Circulating EPCs were also decreased after GroEL1 administration in C57BL/B6 mice. Additionally, GroEL1 inhibited the expression of CD34 and eNOS in C57BL/B6 ischemic muscle. In vitro, GroEL1 impaired the capacity of differentiation, mobilization, tube formation, and migration of EPCs. GroEL1 increased senescence, which was mediated by caspases, p38 MAPK, and ERK1/2 signaling in EPCs. Furthermore, GroEL1 decreased integrin and E-selectin expression and induced inflammatory responses in EPCs. In conclusion, these findings suggest that TLR4 and impaired NO-related mechanisms could contribute to the reduced number and functional activity of EPCs in the presence of GroEL1 from C. pneumoniae.     

Rosuvastatin stimulates clonogenic potential and anti‐inflammatory properties of endothelial progenitor cells

EPCs (endothelial progenitor cells) exert vasculoprotective effects and can be used for regenerative therapies. However, several isolation protocols have been described, with inconsistent results. Statins are among the most effective compounds that stimulate EPC numbers in vivo and ex vivo. We aim to describe the effects of rosuvastatin on different subtypes of putative EPCs. EPCs were cultured from mononuclear cells of blood donors and isolated according to three protocols: CFU‐EC (colony forming units‐endothelial cells), early (or ‘monocytic’) EPCs and late outgrown EPCs. Rosuvastatin (0.1–100 nM) was added at the beginning of culture (T0) or after the initial adhesion step (T1). Polarization of monocytic EPCs was assessed as expression of proinflammatory M1 markers (CD68 and CCR2) or anti‐inflammatory M2 markers (CX3CR1, CD163, CD206). We found that 1 nM rosuvastatin increased the number of CFU‐EC and late EPCs by about 3‐fold, while lower concentrations had no significant effects. Rosuvastatin (0.1 nM) increased AcLDL⁺Lectin⁺ early EPCs by about 60%, while higher concentrations exerted inhibitory effects on early EPCs. Addition of rosuvastatin at T0 was more effective in stimulating CFU‐EC and early EPCs, while addition at T1 was more effective in stimulating late EPCs. Rosuvastatin had no effects on proliferation rate of CFU‐EC, early EPCs and late EPCs. We also found that 0.1 nM rosuvastatin reduced the M1/M2 ratio in early EPCs, which retain monocytic features. In conclusion, we show that rosuvastatin had significant stimulatory effects on EPCs irrespective of the culture protocol. Rosuvastatin also induced anti‐inflammatory polarization of monocytic EPCs.     

Effects of tumour necrosis factor-alpha on activity and nitric oxide synthase of endothelial progenitor cells from peripheral blood

The aim of this investigation was to determine whether tumour necrosis factor-alpha (TNF-α) has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with tumour necrosis factor-α (final concentrations: 0, 10, 20, 50 and 100 mg/l) for 0, 6, 12, 24 and 48 h. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding, by direct fluorescence staining. EPC proliferation and migration were assayed using the MTT assay and modified Boyden chamber assay, respectively. EPC adhesion assay was performed by re-plating those cells on fibronectin-coated dishes, and adherent cells were counted. Tube formation activity was assayed using a tube formation kit. Levels of apoptosis were revealed using an annexin V apoptosis detection kit. Vascular endothelial growth factor Receptor-1 (VEGF-R1) and stromal derived factor-1 (SDF-1) mRNA, assessed by real-time RT-PCR inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were assayed by western blot analysis. Incubation of EPCs with tumour necrosis factor-α reduced EPC proliferation, migration, adhesion, tube formation capacity, iNOS and eNOS in concentration- and time-dependent manners. Tumour necrosis factor-α reduced proliferation, migration, adhesion and tube formation capacity of EPCs. TNF-α increased EPC apoptosis level, reduced VEGF-R1 and SDF-1 mRNA expression; tumour necrosis factor-α also reduced iNOS and eNOS in the EPCs.     

Determine exogenous human DDAH2 gene function in rabbit bone marrow–derived endothelial progenitor cells in vitro

The in vitro amplification of endothelial progenitor cells (EPCs) is an important method because of its role in gene transferring and regenerative medicine. In this study, we isolated rabbit bone marrow–derived EPCs to further manipulation and overexpression of dimethylarginine dimethylaminohydrolase (DDAH) in EPCs. Isolated EPCs were cultured, expanded in endothelial basal medium. Morphology of EPCs and expression levels of surface markers detected using immunocytochemistry staining and through the use of flow cytometery. Endothelial progenitor cells were transfected with plasmid vectors expressing human DDAH2 (DDAH2‐EPCs). Three days after gene transfer, positive transfected‐EPCs proliferation and DDAH activity were assayed. We observed colonies conformation and endothelium‐like morphology gradually in the third week of culture. Characterization results revealed positive expression of EPC surface markers CD106, Flk‐1, vWF, and CD34 using few identification techniques. Overexpression of DDAH2 increased citrulline production after 96 hours of transfection, 235.34 ± 0.69 vs 95.26 ± 5.76 ng/mL; P = .023. These results suggest that cell population with EPC characteristics can be simply isolated from rabbit bone marrow and successfully engineered to overexpress exogenous gene. In this study, we offer a feasible method to isolate and identify EPCs from bone marrow. In addition, an efficient transfection with a plasmid vector (without risk of interference) can be constructed a hybrid structure with EPC and DDAH2 gene to examine their function in vitro.     

Pre-procedural atorvastatin mobilizes endothelial progenitor cells: clues to the salutary effects of statins on healing of stented human arteries

Objectives

Recent clinical trials suggest an LDL-independent superiority of intensive statin therapy in reducing target vessel revascularization and peri-procedural myocardial infarctions in patients who undergo percutaneous coronary interventions (PCI). While animal studies demonstrate that statins mobilize endothelial progenitor cells (EPCs) which can enhance arterial repair and attenuate neointimal formation, the precise explanation for the clinical PCI benefits of high dose statin therapy remain elusive. Thus we serially assessed patients undergoing PCI to test the hypothesis that high dose Atorvastatin therapy initiated prior to PCI mobilizes EPCs that may be capable of enhancing arterial repair.

Methods and Results

Statin naïve male patients undergoing angiography for stent placement were randomized to standard therapy without Atorvastatin (n = 10) or treatment with Atorvastatin 80 mg (n = 10) beginning three days prior to stent implantation. EPCs were defined by flow cytometry (e.g., surface marker profile of CD45dim/34+/133+/117+). As well, we also enumerated cultured angiogenic cells (CACs) by standard in vitro culture assay. While EPC levels did not fluctuate over time for the patients free of Atorvastatin, there was a 3.5-fold increase in EPC levels with high dose Atorvastatin beginning within 3 days of the first dose (and immediately pre-PCI) which persisted at 4 and 24 hours post-PCI (p<0.05). There was a similar rise in CAC levels as assessed by in vitro culture. CACs cultured in the presence of Atorvastatin failed to show augmented survival or VEGF secretion but displayed a 2-fold increase in adhesion to stent struts (p<0.05).

Conclusions

High dose Atorvastatin therapy pre-PCI improves EPC number and CAC number and function in humans which may in part explain the benefit in clinical outcomes seen in patients undergoing coronary interventions.     

Aging, exercise, and endothelial progenitor cell clonogenic and migratory capacity in men.

Numerical and functional impairment of circulating endothelial progenitor cells (EPCs) is thought to contribute to vascular aging and the associated increase in cardiovascular risk. We tested the following hypotheses: 1) EPC clonogenic and migratory capacity decrease progressively with age in healthy, sedentary adult men; and 2) regular aerobic exercise will improve EPC clonogenic and migratory capacity in previously sedentary middle-aged and older men. Peripheral blood samples were collected from 46 healthy sedentary men: 10 young (26 +/- 1 yr), 15 middle-aged (47 +/- 1 yr), and 21 older (63 +/- 1 yr). Mononuclear cells were isolated and preplated for 2 days, and nonadherent cells were further cultured for 7 days to determine EPC colony-forming units. Migratory activity of EPCs was determined using a modified Boyden chamber. Ten sedentary middle-aged and older men (59 +/- 3 yr) were studied before and after a 3-mo aerobic exercise intervention. The number of EPC colony-forming units was approximately 75% lower (P < 0.01) in middle-aged (12 +/- 3) and older (8 +/- 2) compared with young (40 +/- 7) men. There was no difference in colony count between middle-aged and older men. EPC migration (fluorescent units) was significantly reduced in older (453 +/- 72) compared with young (813 +/- 114) and middle-aged (760 +/- 114) men. The exercise intervention increased (P < 0.05) both EPC colony-forming units (10 +/- 3 to 22 +/- 5) and migratory activity (683 +/- 96 to 1,022 +/- 123) in previously sedentary middle-aged and older men. These results provide further evidence that aging adversely affects EPC function. Regular aerobic-endurance exercise, however, is an effective lifestyle intervention strategy for improving EPC clonogenic and migratory capacity in middle-aged and older healthy men.