Properties Of Endothelial Progenitor Cells Research Articles

Improvement of erectile dysfunction using endothelial progenitor cells from fetal cerebral vasculature in the cavernous nerve injury of rats

BackgroundBecause of limited differentiation to endothelium from mesenchymal stem cells, it has been strongly recommended to use endothelial progenitor cells for the regeneration of the damaged endothelium of corpora cavernosa. This study was performed to investigate the immortalized human cerebral endothelial cells and their capability for repairing erectile dysfunction in a rat model of cavernous nerve injury. Circulating endothelial progenitor cells were isolated from human fetal brain vasculature at the periventricular region of telencephalic tissues. Over 95% of CD 31-positive cells were sorted and cultured for 10 days. Human cerebral endothelial progenitor cells were injected into the cavernosa of rats with cavernous nerve injury. Erectile response was then assessed. In in vivo assays, rats were divided into three groups: group 1, sham operation: group 2, bilateral cavernous nerve injury: and group 3, treatment with human cerebral endothelial cells after cavernous nerve injury.ResultsEstablished immortalized circulating endothelial progenitor cells showed expression of human telomerase reverse transcriptase transcript by RT-PCR. They also showed the expression of vascular endothelial growth factor, von Willebrand factor, vascular endothelial growth factor receptor, and CD31, cell type-specific markers for endothelial cells by RT-PCR. In in vitro angiogenesis assays, they demonstrated tube formation that suggested morphological properties of endothelial progenitor cells. In in vivo assays, impaired erectile function of rat with cavernous nerve injury recovered at 2, 4, and 12 weeks after transplantation of human cerebral endothelial cells into the cavernosa.ConclusionsTelomerase reverse transcriptase-circulating endothelial progenitor cells from fetal brain vasculature could repair erectile dysfunction of rats with cavernous nerve injury.

"Endothelial Antibody Factory" at the Blood Brain Barrier: Novel Approach to Therapy of Neurodegenerative Diseases.

The failures of anti-β-amyloid immunotherapies suggested that the very low fraction of injected antibodies reaching the brain parenchyma due to the filtering effect of the BBB may be a reason for the lack of therapeutic effect. However, there is no treatment, as yet, for the amyotrophic lateral sclerosis (ALS) despite substantial evidence existing of the involvement of TDP-43 protein in the evolution of ALS. To circumvent this filtering effect, we have developed a novel approach to facilitate the penetration of antibody fragments (Fabs) into the brain parenchyma. Leveraging the homing properties of endothelial progenitor cells (EPCs), we transfected, ex vivo, such cells with vectors encoding anti-β-amyloid and anti-TDP43 Fabs turning them into an “antibody fragment factory”. When injected these cells integrate into the BBB, where they secrete anti-TDP43 Fabs. The results showed the formation of tight junctions between the injected engineered EPCs and the unlabeled resident endothelial cells. When the EPCs were further modified to express the anti-TDP43 Fab, we could observe integration of these cells into the vasculature and the secretion of Fabs. Results confirm that production and secretion of Fabs at the BBB level leads to their migration to the brain parenchyma where they might exert a therapeutic effect.

Interleukin-10 Deficiency Alters Endothelial Progenitor Cell-Derived Exosome Reparative Effect on Myocardial Repair via Integrin-Linked Kinase Enrichment.

Rationale: Systemic inflammation compromises the reparative properties of endothelial progenitor cell (EPC) and their exosomes on myocardial repair, although the underlying mechanism of loss of function of exosomes from inflamed EPCs is still obscure. Objective: To determine the mechanisms of IL-10 (interleukin-10) deficient-EPC-derived exosome dysfunction in myocardial repair and to investigate if modification of specific exosome cargo can rescue reparative activity. Methods and Results: Using IL-10 knockout mice mimicking systemic inflammation condition, we compared therapeutic effect and protein cargo of exosomes isolated from wild-type EPC and IL-10 knockout EPC. In a mouse model of myocardial infarction (MI), wild-type EPC-derived exosome treatment significantly improved left ventricle cardiac function, inhibited cell apoptosis, reduced MI scar size, and promoted post-MI neovascularization, whereas IL-10 knockout EPC-derived exosome treatment showed diminished and opposite effects. Mass spectrometry analysis revealed wild-type EPC-derived exosome and IL-10 knockout EPC-derived exosome contain different protein expression pattern. Among differentially expressed proteins, ILK (integrin-linked kinase) was highly enriched in both IL-10 knockout EPC-derived exosome as well as TNFα (tumor necrosis factor-α)-treated mouse cardiac endothelial cell-derived exosomes (TNFα inflamed mouse cardiac endothelial cell-derived exosome). ILK-enriched exosomes activated NF-κB (nuclear factor κB) pathway and NF-κB-dependent gene transcription in recipient endothelial cells and this effect was partly attenuated through ILK knockdown in exosomes. Intriguingly, ILK knockdown in IL-10 knockout EPC-derived exosome significantly rescued their reparative dysfunction in myocardial repair, improved left ventricle cardiac function, reduced MI scar size, and enhanced post-MI neovascularization in MI mouse model. Conclusions: IL-10 deficiency/inflammation alters EPC-derived exosome function, content and therapeutic effect on myocardial repair by upregulating ILK enrichment in exosomes, and ILK-mediated activation of NF-κB pathway in recipient cells, whereas ILK knockdown in exosomes attenuates NF-κB activation and reduces inflammatory response. Our study provides new understanding of how inflammation may alter stem cell-exosome-mediated cardiac repair and identifies ILK as a target kinase for improving progenitor cell exosome-based cardiac therapies.

Endoglin as an Adhesion Molecule in Mature and Progenitor Endothelial Cells: A Function Beyond TGF-β.

Endoglin (ENG) is a transmembrane glycoprotein expressed on endothelial cells that functions as a co-receptor for several ligands of the transforming growth factor beta (TGF-β) family. ENG is also a recognized marker of angiogenesis and mutations in the endoglin gene are responsible for Hereditary Hemorrhagic Telangiectasia (HHT) type 1, a vascular disease characterized by defective angiogenesis, arteriovenous malformations, telangiectasia, and epistaxis. In addition to its involvement in the TGF-β family signaling pathways, several lines of evidence suggest that the extracellular domain of ENG has a role in integrin-mediated cell adhesion via its RGD motif. Indeed, we have described a role for endothelial ENG in leukocyte trafficking and extravasation via its binding to leukocyte integrins. We have also found that ENG is involved in vasculogenic properties of endothelial progenitor cells known as endothelial colony forming cells (ECFCs). Moreover, the binding of endothelial ENG to platelet integrins regulate the resistance to shear during platelet-endothelium interactions under inflammatory conditions. Because of the need for more effective treatments in HHT and the involvement of ENG in angiogenesis, current studies are aimed at identifying novel biological functions of ENG which could serve as a therapeutic target. This review focuses on the interaction between ENG and integrins with the aim to better understand the role of this protein in blood vessel formation driven by progenitor and mature endothelial cells.

Synergic effects of VEGF-A and SDF-1 on the angiogenic properties of endothelial progenitor cells.

Here we investigated the impact of hypoxic environment on the angiogenic properties of early-outgrowth endothelial progenitor cells (EPCs), with particular focus on the role of secreted vascular endothelial growth factor-A (VEGF-A) and stromal derived factor-1 (SDF-1) in mediating these effects. We found that cultured EPCs secreted factors with paracrine effects on chemotaxis, migration, proliferation and tube formation of mature endothelial cells (ECs), and these properties were not affected by hypoxia. Depletion of VEGF-A did not change the ability of EPC-conditioned medium (CM) to promote EC migration and tube formation in vitro, suggesting that the pro-angiogenic paracrine effects of EPCs did not totally rely on the presence of VEGF-A. These findings were confirmed by in vivo experiments, on a mouse model of hind limb ischaemia, which showed that VEGF-depleted EPC-CM sustained tissue perfusion at the same level as complete EPC-CM. However, concomitant deletion of VEGF-A and SDF-1 in EPC-CM impaired the pro-angiogenic properties of EPC-CM, by inhibition of EC spreading in culture, tube-like structure formation on Matrigel support, in vivo neovessels formation and ischaemic hind limb regeneration. Taken together, our data demonstrate that: (i) hypoxia does not affect the capacity of EPCs to support the angiogenic process; (ii) the absence of either VEGF-A or SDF-1 from EPC-CM can be rescued by the presence of the other one, so that the overall angiogenic effects remain unchanged; and (iii) and the concomitant deletion of VEGF-A and SDF-1 from EPC-CM impairs its pro-angiogenic effect, both in vitro and in vivo. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of mesenchymal stem cell-derived cytokines on the functional properties of endothelial progenitor cells

Human mesenchymal stem cell (hMSC) is a potential source for cell therapy due to its property to promote tissue repair. Although, it has been known that hMSCs promote tissue repair via angiogenic cytokines, the interaction between hMSC-derived cytokines and the endothelial progenitor cells (EPCs), which play an important role in tissue neovascularization, is poorly characterized. We investigate the effect of cytokine released from different sources of hMSCs including bone marrow and gestational tissues on the EPC functions in vitro. The migration, extracellular matrix invasion and vessel formation of EPCs were studied in the presence or absence of cytokines released from various sources of hMSCs using transwell culture system. The migration of EPCs was highest when co-culture with secretory factors from placenta-derived hMSCs (PL-hMSCs) compared to those co-culture with other sources of hMSCs. For invasion and vessel formation, secretory factors from bone marrow-derived hMSCs (BM-hMSCs) could produce the maximal enhancement compared to other sources. We further identified the secreted cytokines and found that the migratory-enhancing cytokine from PL-hMSCs was PDGF-BB while the enhancing cytokine from BM-hMSCs on invasion was IGF-1. For vessel formation, the cytokines released from BM-hMSCs were IGF1 and SDF-1. In conclusion, hMSCs can release angiogenic cytokines which increase the migration, invasion and vessel forming capacity of EPCs. We can then use hMSCs as a source of angiogenic cytokines to induce neovascularization in injured/ischemic tissues.

Endothelial progenitor cells derived from the peripheral blood of halfpipe- snowboarding athletes display specific functional properties.

In this study, we compared the functional properties of endothelial progenitor cells (EPCs) derived from halfpipe-snowboarding athletes who train under hyperoxic conditions with those derived from normal subjects who lived under normoxic conditions. Peripheral blood-derived EPCs were isolated from both halfpipe-snowboarding athletes and normal humans. Cellular growth dynamics, lipoprotein transport, and gene expression of cultured EPCs were compared between the two groups of cells. Results indicate that cytoactivity of EPCs from athletes was higher than that of EPCs from control subjects. This study suggests that function of EPCs from snowboarding athletes may be better than that of EPCs from normal humans, which demonstrates the benefits of training under hyperoxic conditions.

Endothelial progenitor cell levels in juvenile idiopathic arthritis patients: effects of anti-inflammatory therapies.

BackgroundJuvenile idiopathic arthritis (JIA), similarly to other arthritides, can be associated with damage of endothelial layer of which structure and function is dependent on reparative properties of endothelial progenitor cells (EPC). To date, it remained unknown whether EPC numbers are altered in young JIA patients and whether on-going anti-inflammatory therapies could exert positive effects on these progenitor cells.MethodsWe performed a quantitative analysis of EPC numbers in 25 patients diagnosed with JIA according to International League of Associations for Rheumatism (ILAR) criteria [age 11.50 (7.50-15.00) years] in a broad context of inflammatory and cardiovascular parameters as well as different types of anti-inflammatory treatments. 11 healthy children [age 13.00 (11.00-14.00) years] were recruited as a control group.ResultsWe demonstrated that EPC numbers were similar in JIA patients and control subjects (0.02% vs. 0.05%, respectively, p = 0.37). EPC levels in JIA patients were negatively correlated with index of insulin resistance (rho = -0.458, p = 0.021), endogenous insulin (rho = -0.472, p = 0.017), triglyceride (rho = -0.438, p = 0.029) and TNF-alpha levels (rho = -0.446, p = 0.026). Notably, glucocorticoid (GC) therapy, was associated with detection of decreased EPC levels in JIA patients (p = 0.023). In contrast, methothrexate (MTX) and etanercept therapy in JIA patients did not affect EPC levels (p = 0.92 and p = 0.08, respectively).ConclusionsWe found that EPC numbers are maintained at normal levels in JIA patients and are not enhanced by disease-specific anti-inflammatory treatments.

Prohealing Endothelial Progenitor Cell Capture Stents

Since the early days of percutaneous transluminal angioplasty, it has been recognized that balloon inflation disrupts the endothelial monolayer and injures normal arterial segments.1 This initiates a repair process that is mediated by platelets, inflammatory cells, and circulating progenitor cells that are recruited to sites of injury. There has been recent interest in the prohealing properties of endothelial progenitor cells (EPCs). Early studies identified these cells by expression of the surface antigen CD34 and demonstrated that they promoted reendothelialization after balloon injury.2,3 Because of the fact that these cells are relatively rare in the circulation (0.005%–0.01% of the total white blood cell count), it is not surprising that a stent was designed to capture circulating EPCs and sequester the cells to promote rapid reendothelialization, to decrease thrombogenicity, and to prevent restenosis.4 To accomplish this, the stent incorporated a monoclonal CD34 antibody in a proprietary polysaccharide intermediate coating that was adhered to a stainless steel stent; in 2010, the stent platform was changed to cobalt chromium.5 The safety and efficacy of the EPC capture stent have been studied extensively in clinical registries and randomized trials. The Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth (HEALING) registries established a safety profile for the stent but demonstrated what would become a common theme; the stent did not decrease late lumen loss (LLL) to the same degree observed for drug-eluting stents (DES). This finding was attributed initially to variation in the number of circulating EPCs because of differences in the use …

Alcohol Consumption Negates Estrogen-mediated Myocardial Repair in Ovariectomized Mice by Inhibiting Endothelial Progenitor Cell Mobilization and Function

We have shown previously that estrogen (estradiol, E2) supplementation enhances voluntary alcohol consumption in ovariectomized female rodents and that increased alcohol consumption impairs ischemic hind limb vascular repair. However, the effect of E2-induced alcohol consumption on post-infarct myocardial repair and on the phenotypic/functional properties of endothelial progenitor cells (EPCs) is not known. Additionally, the molecular signaling of alcohol-estrogen interactions remains to be elucidated. This study examined the effect of E2-induced increases in ethanol consumption on post-infarct myocardial function/repair. Ovariectomized female mice, implanted with 17β-E2 or placebo pellets were given access to alcohol for 6 weeks and subjected to acute myocardial infarction. Left ventricular functions were consistently depressed in mice consuming ethanol compared with those receiving only E2. Alcohol-consuming mice also displayed significantly increased infarct size and reduced capillary density. Ethanol consumption also reduced E2-induced mobilization and homing of EPCs to injured myocardium compared with the E2-alone group. In vitro, exposure of EPCs to ethanol suppressed E2-induced proliferation, survival, and migration and markedly altered E2-induced estrogen receptor-dependent cell survival signaling and gene expression. Furthermore, ethanol-mediated suppression of EPC biology was endothelial nitric oxide synthase-dependent because endothelial nitric oxide synthase-null mice displayed an exaggerated response to post-acute myocardial infarction left ventricular functions. These data suggest that E2 modulation of alcohol consumption, and the ensuing EPC dysfunction, may negatively compete with the beneficial effects of estrogen on post-infarct myocardial repair.

A switch toward angiostatic gene expression impairs the angiogenic properties of endothelial progenitor cells in low birth weight preterm infants

A switch toward angiostatic gene expression impairs the angiogenic properties of endothelial progenitor cells in low birth weight preterm infants

Phenotypic and functional characterization of endothelial progenitor cells isolated from peripheral blood of renal cell carcinoma patients

Endothelial progenitor cells (EPCs) are mobilized from either bone marrow or arterial walls to restore blood perfusion to ischemic organs and establish the vascular network within growing tumors [1]. The Ca2+ machinery plays a key role in EPC activation and might serve a molecular target for novel therapies of highly angiogenic tumors, such as renal cell carcinoma (RCC) [1]. The Ca2+ toolkit is remodelled in EPCs isolated from RCC patients (RCC-EPCs) as respect to healthy donors [2]. The present study was undertaken to evaluate for the first time the functional properties of EPCs isolated from tumor patients by focusing on RCC-EPCs. We extended our analysis at microscopic level by monitoring the sub-cellular structure of RCC-EPCs relative to their Ca2+ signalling fingerprint. Our results showed a striking functional and ultrastructural difference between RCC-EPCs and their normal counterparts, which might be the basis for designing novel, more specific anti-angiogenic treatments.

Towards the therapeutic use of vascular smooth muscle progenitor cells

Recent advances in the development of alternative proangiogenic and revascularization processes, including recombinant protein delivery, gene therapy, and cell therapy, hold the promise of greater efficacy in the management of cardiovascular disease in the coming years. In particular, vascular progenitor cell-based strategies have emerged as an efficient treatment approach to promote vessel formation and repair and to improve tissue perfusion. During the past decade, considerable progress has been achieved in understanding therapeutic properties of endothelial progenitor cells, while the therapeutic potential of vascular smooth muscle progenitor cells (SMPC) has only recently been explored; the number of the circulating SMPC being correlated with cardiovascular health. Several endogenous SMPC populations with varying phenotypes have been identified and characterized in the peripheral blood, bone marrow, and vascular wall. While the phenotypic entity of vascular SMPC is not fully defined and remains an evolving area of research, SMPC are increasingly recognized to play a special role in cardiovascular biology. In this review, we describe the current approaches used to define vascular SMPC. We further summarize the data on phenotype and functional properties of SMPC from various sources in adults. Finally, we discuss the role of SMPC in cardiovascular disease, including the contribution of SMPC to intimal proliferation, angiogenesis, and atherosclerotic plaque instability as well as the benefits resulting from the therapeutic use of SMPC.

A switch toward angiostatic gene expression impairs the angiogenic properties of endothelial progenitor cells in low birth weight preterm infants

AbstractLow birth weight (LBW) is associated with increased risk of cardiovascular diseases at adulthood. Nevertheless, the impact of LBW on the endothelium is not clearly established. We investigate whether LBW alters the angiogenic properties of cord blood endothelial colony forming cells (LBW-ECFCs) in 25 preterm neonates compared with 25 term neonates (CT-ECFCs). We observed that LBW decreased the number of colonies formed by ECFCs and delayed the time of appearance of their clonal progeny. LBW dramatically reduced LBW-ECFC capacity to form sprouts and tubes, to migrate and to proliferate in vitro. The angiogenic defect of LBW-ECFCs was confirmed in vivo by their inability to form robust capillary networks in Matrigel plugs injected in nu/nu mice. Gene profile analysis of LBW-ECFCs demonstrated an increased expression of antiangiogenic genes. Among them, thrombospondin 1 (THBS1) was highly expressed at RNA and protein levels in LBW-ECFCs. Silencing THBS1 restored the angiogenic properties of LBW-ECFCs by increasing AKT phosphorylation. The imbalance toward an angiostatic state provide a mechanistic link between LBW and the impaired angiogenic properties of ECFCs and allows the identification of THBS1 as a novel player in LBW-ECFC defect, opening new perspectives for novel deprogramming agents.

Improved angiogenesis by P-selectin glycoprotein ligand-1 overexpression in endothelial progenitor cells

To explore whether overexpression of P-selectin glycoprotein ligand-1 (PSGL-1) promotes the adhesive ability of endothelial progenitor cells and functionally facilitates neovascularization in mouse model of hindlimb ischemia. Rat endothelial progenitor cells were transfected with recombinant adenovirus vector encoding human PSGL-1. The mRNA and protein expression levels of PSGL-1 were measured by RT-PCR and Western blot, respectively. The effect of overexpression of PSGL-1 in endothelial progenitor cells was analyzed by adherence assay. Histological examination of skeletal muscle sections retrieved from the mouse ischemic hindlimbs was performed, and the hindlimb blood flow was measured by laser Doppler flow meter. Adenovirus vector expressing of PSGL-1 gene was successfully constructed with high titer of 3.1 × 10¹¹ pfu/ml. After transfection, PSGL-1 gene was highly expressed in the transfected endothelial progenitor cells. In vitro assay showed that overexpression of PSGL-1 enhanced the adhesive properties of endothelial progenitor cells. When the transfected endothelial progenitor cells were transplanted into the ischemic hindlimb of nude mice, the number of new capillary vessels was (41.0 ± 2.2)/HPF compared to that of (21.0 ± 2.5)/HPF in the negative control group and (10.0 ± 1.6)/HPF in the blank control group (P < 0.01). Furthermore, the blood flow was increased in the experimental group (119.1% ± 7.0%), whereas in the negative control group, it was (93.3% ± 3.0%) and in the blank control group it was (76.3% ± 12.0%), P < 0.01. Overexpression of PSGL-1 enhances the adhesive and angiogenic properties of endothelial progenitor cells. The approach may provide an effective therapeutic strategy to improve the efficiency of cell-based proangiogenic therapy.

Exposure to platelets promotes functional properties of endothelial progenitor cells

Recent evidence indicates that endothelial progenitor cells (EPCs) have an important role in the process of repair following vascular injury, and that platelets mediate their recruitment to sites of injury. Platelets and EPCs can interact and bind directly. However, there is limited information on the effect of platelets on EPC function following this interaction. We, therefore, aimed to assess the in vitro effect of platelets on functional properties of EPCs. Human EPCs were isolated from donated Buffy coats and purified on a magnetic separation column specific for CD133. They were incubated either on fibronectin matrix, or co-incubated with washed platelets (isolated from healthy volunteers), for 7 days. Number of EPC colony forming units (CFU) was quantified, and endothelial cell lineage confirmed by immunostaining. Functional properties of the cultured cells were evaluated by MTT--proliferation assay and migration assay using the Boyden chamber. Co-incubation of EPCs with platelets compared to incubation of EPCs alone (on fibronectin matrix) resulted in higher number of CFUs after 7 days (6.5 ± 1.3 vs. 3.5 ± 0.5 CFUs/well, respectively, P = 0.005). In addition, co-incubation of EPCs with platelets versus EPCs alone was associated with higher proportion of living cells, by the MTT assay (0.2 ± 0.01 vs. 0.12 ± 0.04 MTT 570 nm respectively, P = 0.003), and higher number of migrated EPCs, assessed by the migration assay (1400 ± 212 vs. 580 ± 180 migrated cells/2000 cells, respectively, P < 0.0001). In vitro exposure to platelets promotes the capacity of EPCs to form colonies, proliferate and migrate. Therefore, the interaction with platelets appears to augment EPC functional properties.

Overexpression of P-selectin glycoprotein ligand-1 enhances adhesive properties of endothelial progenitor cells through Syk activation

P-selectin glycoprotein ligand-1 (PSGL-1) not only functions as an anchor molecule to capture monocytes and other leukocytes to endothelial cells in ischemic tissue by its interaction with P-selectin, but also transduces signals to initiate firm adhesion. Endothelial progenitor cells are derived from monocytes and play a very important role in neovascularization. Transplantation of endothelial progenitor cells is a promising therapeutic strategy to improve treatment of ischemic disease such as myocardial and cerebral infarction; however, its efficacy is now limited by the fact that few of the transplanted cells adhere to and accumulate in the ischemic tissue. In this study we aimed to investigate whether the overexpression of PSGL-1 gene promotes endothelial progenitor cells adhesion activity and explore the underlying mechanisms. We found that after transfection with human PSGL-1 gene, endothelial progenitor cells exhibited higher affinity to activated human umbilical vein endothelial cells or recombined P-selectin/ICAM-1 monolayer. The overexpression of PSGL-1-enhanced beta2-integrin expression on endothelial progenitor cells surface, and this effect was Syk dependent. The specific Syk inhibitor abolished the elevating effect of overexpression of PSGL-1 on surface beta2-integrin expression and the adhesive affinity of endothelial progenitor cells. These results suggested that Syk plays a key role in signal transduction downstream of PSGL-1 in endothelial progenitor cells, and the overexpression of PSGL-1 improves endothelial progenitor cells adhesive properties through enhanced activation of Syk and following integrin activation.

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.

Enhanced adhesive properties of endothelial progenitor cells (EPCs) in patients with SLE

Endothelial progenitor cells (EPCs) are a population of bone marrow-derived cells present in the peripheral circulation, which possess the ability to migrate into areas where angioneogenesis is required and differentiate upon adhesion into mature endothelial cells. EPCs have reparative properties, are able to combat ischemia and have previously been shown to be decreased in level and function in inflammatory conditions. Systemic lupus erythematosus (SLE) is a multi-organ autoimmune inflammatory disorder associated with significantly increased cardiovascular morbidity and mortality. To investigate the numbers and functional properties of EPCs among patients suffering from SLE, thirty-one patients suffering from active SLE (American College of Rheumatology criteria) as well as 54 healthy controls were recruited. Disease activity was assessed using the SLEDAI score. Peripheral blood mononuclear cells were isolated and EPC numbers evaluated by the colony-forming unit (CFU) method. Functional properties were evaluated by EPC adherence to fibronectin. No significant difference was found between numbers of circulating EPC colony-forming units (CFUs) among patients with SLE and healthy individuals. A significant increase in adhesive capacity of EPCs to immobilized fibronectin was evident in patients with SLE compared to controls. An increase in adhesive capacity of circulating EPCs was observed in patients with SLE which may be related to altered endothelial function.

Genetic engineering with endothelial nitric oxide synthase improves functional properties of endothelial progenitor cells from patients with coronary artery disease: an in vitro study

Recent studies have reported a marked impairment in the number and functions of endothelial progenitor cells (EPCs) in patients with coronary artery disease (CAD). In view of an important role of eNOS in angiogenesis, in the present study, we evaluated the effects of eNOS gene transfer in ex vivo expanded EPCs isolated from patients with CAD. The expanded EPCs were transfected with mammalian expression vector pcDNA3.1-eNOS containing the full-length human eNOS gene using lipofectamine. About 35-40% of the eNOS-EPCs had higher expression of eNOS as compared to untransfected EPCs. EPCs transfected with pcDNA3.0-EGFP, the plasmid vector expressing green fluorescent protein (GFP) were used as control. The untransfected, GFP-transfected and eNOS-transfected EPCs were compared in terms of important functional attributes of angiogenesis such as proliferation, migration, differentiation and adhesion/integration into tube-like structures in vitro. Functional studies revealed that in the presence of defined growth conditions, compared to the untransfected and GFP-transfected cells, eNOS-EPCs from patients with CAD have a significant increase in [3H] thymidine-labeled DNA (P < 0.01), migration (14.6 +/- 1.8 and 16.5 +/- 1.9 vs. 23.5 +/- 3.4 cells/field, P < 0.01), ability to differentiate into endothelial-like spindle-shaped cells (46 +/- 4.5 and 56.5 +/- 2.1 vs. 93.2 +/- 6.6 cells/field, P < 0.001) and also incorporation into tube-like structures on the matrigel (GFP-EPCs: 21.25 +/- 2.9 vs. GFP-eNOS-EPCs: 34.5 +/- 5.5 cells/field, P < 0.05). We conclude that eNOS gene transfection is a valuable approach to augment angiogenic properties of ex vivo expanded EPCs and eNOS-modified EPCs may offer significant advantages than EPCs alone in terms of their clinical use in patients with myocardial ischemia.