Outgrowth Endothelial Progenitor Cells Research Articles

Functional late outgrowth endothelial progenitors isolated from peripheral blood of burned patients

BackgroundBioengineered skin substitutes are increasingly considered as a useful option for the treatment of full thickness burn injury. Their viability following grafting can be enhanced by seeding the skin substitute with late outgrowth endothelial progenitor cells (EPCs). However, it is not known whether autologous EPCs can be obtained from burned patients shortly after injury. MethodsLate outgrowth EPCs were isolated from peripheral blood sampled obtained from 10 burned patients (extent 19.6±10.3% TBSA) within the first 24h of hospital admission, and from 7 healthy subjects. Late outgrowth EPCs were phenotyped in vitro. ResultsIn comparison with similar cells obtained from healthy subjects, growing colonies from burned patients yielded a higher percentage of EPC clones (46 versus 17%, p=0.013). Furthermore, EPCs from burned patients secreted more vascular endothelial growth factor (VEGF) into the culture medium than did their counterparts from healthy subjects (85.8±56.2 versus 17.6±14pg/mg protein, p=0.018). When injected to athymic nude mice 6h after unilateral ligation of the femoral artery, EPCs from both groups of subjects greatly accelerated the reperfusion of the ischaemic hindlimb and increased the number of vascular smooth muscle cells. ConclusionsThe present study supports that, in patients with burns of moderate extension, it is feasible to obtain functional autologous late outgrowth EPCs from peripheral blood. These results constitute a strong incentive to pursue approaches based on using autotransplantation of these cells to improve the therapy of full thickness burns.

008 Microrna Expression in Blood Derived Mononuclear Cells During Maturation From Early to Late Outgrowth Endothelial Progenitor Cells

Endothelial Progenitor Cells (EPCs) circulate in the peripheral blood mononuclear cell (PBMC) fraction and can be selected under culture conditions which include appropriate matrix components and endothelial growth factors. Early outgrowth EPCs appear after the first 3 to 5 days of selective culture, and these nonproliferative cells still express MNC markers (i.e. CD14 and CD45), but also express some endothelial cell (EC) markers including CD31 and VEGFR2. Late outgrowth EPCs appear after 2 weeks, and exhibit a strong EC phenotype, and this highly proliferative population has lost all leukocyte markers. MicroRNAs (miRNAs) are small noncoding RNAs that have emerged as important regulators of gene expression. The aim of this study was to define the changes in miRNA expression profiles in MNC cultures during evolution from early to late outgrowth EPC. Changes in the expression of specific miRNAs contribute to the endothelial specification of cultured MNCs and the emergence of early or late outgrowth EPCs. Mononuclear cells (MNCs) were isolated from healthy participants (n=4) by leukaphesis and cultured for 1, 3, 5, 7 and 9 days under conditions promoting endothelial differentiation. Late outgrowth EPCs were derived from the same cultures and appeared after 14-21 days (n=3). MiRNA expression was assessed by (q)RT-PCR (Taqman), using a panel of 13 miRNAs previously identified in PBMCs, EPCs or mature ECs. MiRNA expression profiles were very similar in cells between 1 and 3 days (cultured MNCs); and 5, 7 and 9 days (early outgrowth EPCs), and therefore these were grouped for further analysis. MiR-92a was highly dominant in day 1-3 MNC cultures and remained relatively constant throughout the culture period. In early outgrowth EPCs (day 5-9), the expression of miR-146a increased 2.9±0.04 fold, (p<0.05). In contrast, late outgrowth EPCs exhibited marked increases in expression of miRs-126 , -222 and -221 compared to early EPCs (by 453-, 14- and 8.4-folds respectively; p<0.05). The ratio of miR-126 to miR-146 provided the strongest indicator of endothelial differentiation. These data demonstrate that MNCs exhibit specific patterns of miRNA expression tightly associated with the progression from early to late outgrowth EPCs. MiRNAs may play a critical role in regulating the maturation of MNCs from early to late outgrowth EPCs, and enhancing miR-126 or suppressing miR-146 may facilitate their differentiation to an endothelial phenotype.

416 Inhibition of Four- and- a- Half LIM Domain Protein 2 Increases Survival and Migratory Capacity of Human Early Outgrowth Endothelial Progenitor Cells Through Upregulation of Sphingosin Kinase- 1: Implications for Endothelial Regeneration

Inhibition of Four-and-a-half LIM domain protein-2 (FHL-2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Endothelial progenitor cells (EPCs) substantially contribute to endothelial repair. We investigated the role of FHL-2 in the regulation of early outgrowth EPC number and function. Early outgrowth EPCs were obtained from human peripheral blood. FHL-2 knockdown in EPCs by small-interfering RNA (siRNA) resulted in a significant increase in EPC number and a reduction of apoptosis (by 40%), as indicated by a decrease of cleaved caspase-3, through activation and translocation to the membrane, of sphingosine kinase-1 (SK-1), enzyme that metabolizes sphingosine- 1 phosphate (s1p). Furthermore, FHL-2 siRNA increased significantly (2 fold) stromal derived factor (SDF) -1- induced EPC migration; through upregulation of α-v/β-3 and α-v/β-5 integrins; this was associated with an increase of the F-actin binding protein cortactin, known to promote migration. Interestingly, increased SDF-1- induced EPC migration and upregulation of cortactin by FHL-2 siRNA were totally prevented by CAY10621, a specific inhibitor of SK-1. In addition stimulation of EPCs with exogenous s1p peptide significantly decreased apoptosis and increased SDF-1- induced migration. These results were confirmed In vivo using FHL-2 knockout (FHL-2 −/−) mice. Moreover, apoptosis was significantly decreased and migration increased in endothelial cells exposed to the conditioned medium of FHL-2 −/− vs. WT EPCs. These effects were abolished by VPC23019, an antagonist of sphingosine- 1- phosphate receptor- 1 and 3. Finally, reendothelialization after focal carotid endothelial electric injury in WT mice was significantly increased after application of spleen-derived progenitor cells from FHL-2 −/− mice vs. WT mice. Our findings suggest that FHL-2 negatively regulates early outgrowth EPC function and secretion of paracrine factors. FHL-2 inhibition reduces apoptosis, enhances survival and migratory capacity of EPCs and ECs by upregulating SK-1/s1p pathway, integrin subunits and cortactin; which results in the improvement of endothelial regeneration.

242 EFFECTS OF CHILDHOOD OBESITY ON THE ENDOTHELIAL FUNCTION AND FUNCTIONAL PROPERTIES OF EARLY OUTGROWTH ENDOTHELIAL PROGENITOR CELLS

Background: The prevalence of cardiovascular risk factors has been increasing among children/adolescents in connection to rapidly increased childhood obesity. Given that endothelial dysfunction is an early event in the development of atherosclerosis and endothelial progenitor cells (EPCs) are important in vascular repair, we wanted to investigate the impact of childhood obesity on the endothelial function and functional properties of EPCs. Methods: We examined 20 overweight/obese (BMI: 33.7±6 kg/m2, Age: 19.2±2.1) and 10 normal weight young people (BMI: 21.8±1.7 kg/m2, Age: 18.5±1.4). These subjects were identified in a longitudinal study that began 5 years ago and remained in their original weight group. EPCs were obtained by culturing Ficoll-density isolated peripheral mononuclear cells on fibronectin-coated dishes. The number of early outgrowth EPCs and their adhesive and migratory capacity were analyzed. The endothelial function was assessed using reactive hyperemia (RH) peripheral arterial tonometry (Itamar). Global fitting was used to compare RH-response curves (GraphPad 5). Results: Compared with controls, overweight/obese subjects had lower RH-response (Figure) and lower number of early outgrowth EPCs (0.33±0.16 vs 0.47±0.2 million cells, p=0.039). However, EPCs expanded from overweight/obese had similar adhesive and migratory capacity as controls. The overweight/obese had higher levels of insulin (12.1±6.7 vs 6.9±2.4 mU/L, p=0.02), systolic blood pressure (121±9 vs 109±9 mmHg, p=0.001) and diastolic blood pressure (72±7 vs 63±5 mmHg, p=0.002) than the controls.Conclusion: Childhood obesity impaired endothelial function and reduced the number of early outgrowth EPCs already in young age.

034 Endothelial Nitric Oxide Synthase-Overexpressing Human Early Outgrowth Endothelial Progenitor Cells Decrease Human Coronary Artery Smooth Muscle Cell Migration Through Paracrine Functions

Arterial restenosis, which occurs in up to 20% of angioplasty patients, is characterized by an excessive vascular smooth muscle cell proliferation resulting from the removal of the endothelial cell lining. Circulating endothelial progenitor cells (EPCs) have the ability to re-colonize and repair the damaged vascular endothelium, reducing restenosis. Nitric oxide (NO) contributes to mobilization and functional activity of EPCs, and estrogen has been shown to increase circulating EPC levels and accelerate the reendothelialization process by EPCs in mice. Moreover NO is important for transducing estrogen-dependent signalling and reendothelialization. We hypothesized that overexpressing endothelial nitric oxide synthase (eNOS), or treatment with estrogen, would potentiate the beneficial effects of EPCs in the context of restenosis. We found that native human early outgrowth EPCs (hEPCs) did not have any effect on human coronary artery smooth muscle cell (HCASMC) proliferation and migration In vitro, evaluated by BrdU incorporation and wound scratch assay respectively. In contrast, the NO donor SNAP significantly decreased the proliferation and migration of HCASMCs. Thereafter, hEPCs were either transfected with a human eNOS plasmid or stimulated with 17β-estradiol (E2) prior to being co-cultured with HCASMCs. Total eNOS protein and eNOS phosphorylation levels were increased by 3- to 3.5-fold in eNOS-transfected or E2-stimulated hEPCs, evaluated by western blot. This was associated with a 3-fold increase in NO production, performed by DAF-FM diacetate immunofluorescence (p<0.05). In eNOS-overexpressing hEPCs, enhanced bcl-2/bax ratio and reduced Annexin V/propidium iodide labeling indicated increased survival. Interestingly, we observed a significant (p<0.05) decrease in HCASMC migration when co-cultured with eNOS-overexpressing hEPCs, by 23%, or with E2-stimulated hEPCs, by 56%. However, HCASMC proliferation was not affected by either eNOS-overexpressing or E2-stimulated hEPCs.These results suggest that overexpressing eNOS in hEPCs increases their survival and enhances their capacity to modulate HCASMC migration through paracrine effects.Réseau de thérapie cellulaire et tissulaire Arterial restenosis, which occurs in up to 20% of angioplasty patients, is characterized by an excessive vascular smooth muscle cell proliferation resulting from the removal of the endothelial cell lining. Circulating endothelial progenitor cells (EPCs) have the ability to re-colonize and repair the damaged vascular endothelium, reducing restenosis. Nitric oxide (NO) contributes to mobilization and functional activity of EPCs, and estrogen has been shown to increase circulating EPC levels and accelerate the reendothelialization process by EPCs in mice. Moreover NO is important for transducing estrogen-dependent signalling and reendothelialization. We hypothesized that overexpressing endothelial nitric oxide synthase (eNOS), or treatment with estrogen, would potentiate the beneficial effects of EPCs in the context of restenosis. We found that native human early outgrowth EPCs (hEPCs) did not have any effect on human coronary artery smooth muscle cell (HCASMC) proliferation and migration In vitro, evaluated by BrdU incorporation and wound scratch assay respectively. In contrast, the NO donor SNAP significantly decreased the proliferation and migration of HCASMCs. Thereafter, hEPCs were either transfected with a human eNOS plasmid or stimulated with 17β-estradiol (E2) prior to being co-cultured with HCASMCs. Total eNOS protein and eNOS phosphorylation levels were increased by 3- to 3.5-fold in eNOS-transfected or E2-stimulated hEPCs, evaluated by western blot. This was associated with a 3-fold increase in NO production, performed by DAF-FM diacetate immunofluorescence (p<0.05). In eNOS-overexpressing hEPCs, enhanced bcl-2/bax ratio and reduced Annexin V/propidium iodide labeling indicated increased survival. Interestingly, we observed a significant (p<0.05) decrease in HCASMC migration when co-cultured with eNOS-overexpressing hEPCs, by 23%, or with E2-stimulated hEPCs, by 56%. However, HCASMC proliferation was not affected by either eNOS-overexpressing or E2-stimulated hEPCs. These results suggest that overexpressing eNOS in hEPCs increases their survival and enhances their capacity to modulate HCASMC migration through paracrine effects. Réseau de thérapie cellulaire et tissulaire

Moderate to High Concentrations of High-Density Lipoprotein From Healthy Subjects Paradoxically Impair Human Endothelial Progenitor Cells and Related Angiogenesis by Activating Rho-Associated Kinase Pathways

Recent clinical evidence has failed to demonstrate the benefits of elevation of serum high-density lipoprotein (HDL), suggesting potential loss of protective effects of HDL at high concentrations. This study aimed to investigate the concentration-related effects of HDL on in vitro and in vivo functions of human endothelial progenitor cells (EPCs) and related angiogenesis. Early and late outgrowth EPCs were generated from human circulating mononuclear cells. Oxidized low-density lipoprotein reduced viability of late outgrowth EPCs, which was reversed dose dependently by HDL. In the absence of oxidized low-density lipoprotein, HDL at low concentrations (5-50 μg/mL, equal to 0.5-5 mg/dL in human) enhanced EPC tube formation by activating phosphatidylinositol-3 kinase/Akt/endothelial NO synthase pathways. Moderate to high concentrations (400-800 μg/mL) of HDL paradoxically enhanced EPC senescence and impaired tube formation by activating Rho-associated kinase (ROCK) and inhibiting phosphatidylinositol-3 kinase/Akt and p38 mitogen-activated protein kinase pathways. Rho-associated kinase inhibitors, either Y27632 or statins, prevented high HDL-induced EPC senescence and improved in vitro tube formation, as well as in vivo capacity of angiogenesis of EPCs. While protecting EPCs from the injury of oxidized low-density lipoprotein, moderate to high concentrations of HDL paradoxically impaired EPCs and related angiogenesis in the absence of oxidized low-density lipoprotein by activating Rho-associated kinase pathways, providing mechanistic evidence of potential hazard effects of HDL.

The effects of low-dose Nepsilon-(carboxymethyl)lysine (CML) and Nepsilon-(carboxyethyl)lysine (CEL), two main glycation free adducts considered as potential uremic toxins, on endothelial progenitor cell function

BackgroundPatients with chronic kidney disease (CKD) are at high risk of cardiovascular disease (CVD). Endothelial progenitor cell (EPCs) dysfunction plays a key role in this pathogenesis. Uremic retention toxins have been reported to be in associated with EPC dysfunction. Advanced glycation end-products (AGEs) free adducts, including Nepsilon-(carboxymethyl)lysine (CML) and Nepsilon-(carboxyethyl)lysine (CEL), are formed by physiological proteolysis of AGEs and released into plasma for urinary excretion. They are retained in CKD patients and are considered to be potential uremic toxins. Though AGEs have been demonstrated to impair EPC function in various ways, the effect of AGE free adducts on EPC function has not been studied. Thus, we examined the role of CML and CEL in the regulation of growth-factor-dependent function in cultured human EPCs and the mechanisms by which they may affect EPC function.MethodsLate outgrowth EPCs were incubated with different concentrations of CML or CEL for up to 72 hours. Cell proliferation was determined using WST-1 and BrdU assays. Cell apoptosis was tested with annexin V staining. Migration and tube formation assays were used to evaluate EPC function.ResultsThough CML and CEL were determined to have anti-proliferative effects on EPCs, cells treated with concentrations of CML and CEL in the range found in CKD patients had no observable impairment on migration or tube formation. CML and CEL did not induce EPC apoptosis. The reduced growth response was accompanied by significantly less phosphorylation of mitogen-activated protein kinases (MAPKs).ConclusionsOur study revealed that CML and CEL at uremic concentrations have low biological toxicity when separately tested. The biologic effects of AGE free adducts on the cardiovascular system merit further study.

Ex Vivo Akt/HO-1 Gene Therapy to Human Endothelial Progenitor Cells Enhances Myocardial Infarction Recovery

The aim of this study was to evaluate the overexpression of genes central to cell survival and angiogenesis to enhance the function of human late outgrowth endothelial progenitor cells (EPCs) and their utility for infarct recovery. Ischemic myocardial injury creates a hostile microenvironment, which is characterized by hypoxia, oxidative stress, and inflammation. The infarct microenvironment prevents adhesion, survival, and integration of cell transplants that promote neovascularization. EPCs are dysfunctional as a result of risk factors in cardiovascular patients. Protein kinase B (Akt) and heme-oxygenase-1 (HO-1) are intracellular proteins that play an important role in angiogenesis and cell survival. Late outgrowth EPCs transduced ex vivo with Akt and HO-1 demonstrate improved adhesion to extracellular matrix, improved migration toward human cardiomyocytes, and an improved paracrine profile under stress. Enhanced late outgrowth EPCs reduce the tumor necrosis factor-α (TNF-α) burden both in vitro and in vivo, attenuating nuclear factor-κB (NF-κB) activity and promoting cell survival. Akt and HO-1 enhance late outgrowth EPC neovascularization, resulting in improved cardiac performance and reduced negative remodeling after myocardial infarction in nude mice. Alteration of the infarct microenvironment through gene modification of human late outgrowth EPCs enhances the function and integration of transplanted cells for restoration of cardiac function.

Tie2 lineage deletion of 6 integrin: endothelial and haematopoietic cells in neovascularization

This editorial refers to ‘Tie2-dependent knockout of α6 integrin subunit in mice reduces post-ischaemic angiogenesis’ by C. Bouvard et al ., pp. 39–47, this issue. The metabolic demand of a mature tissue determines the degree of blood and oxygen supply, which normally is controlled by vasoregulation at the level of arteriolar resistance vessels. However, in specific conditions such as inflammation and excessive tissue growth including tumours, the metabolic demand exceeds the supply by autoregulation and therefore requires neovascularization. The need for additional blood supply and neovascularization becomes urgent after an acute vascular occlusion. Rapid formation of collaterals is then required, while the distal hypoxic tissue simultaneously shifts to the production of pro-angiogenic factors, in particular vascular endothelial growth factor, which subsequently induces sprouting angiogenesis in an attempt to connect to other perfused vessels and to restore oxygen and nutrient supply to the ischaemic tissue. In addition, circulating monocytes and endothelial progenitor cells (EPCs) are recruited to the sites of structural arterial widening (arteriogenesis) and sprouting angiogenesis. Monocytes and circulating angiogenic cells (monocyte-lineage cells that acquire several endothelial markers, also called early outgrowth EPCs) support the vascular adaptation in a paracrine manner.1 Furthermore, endothelial colony-forming cells (ECFCs, also called late outgrowth EPCs), which are present in small numbers …

Abstract 200: Inhibition of Four-and-a-Half Lim Domain Protein 2 Increases Survival and Migratory Capacity of Human Early Outgrowth Endothelial Progenitor Cells Through Upregulation of Sphingosine Kinase-1: Implications for Endothelial Regeneration

Background: Inhibition of Four-and-a-half LIM domain protein-2 (FHL-2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Endothelial progenitor cells (EPCs) substantially contribute to endothelial repair. We investigated the role of FHL-2 in the regulation of early outgrowth EPC number and function. Methods and Results: Early outgrowth EPCs were obtained from human peripheral blood. FHL-2 knockdown in EPCs by small-interfering RNA (siRNA) resulted in a significant increase in EPC number and a reduction of apoptosis (by 40%), as indicated by a decrease of cleaved caspase-3, through activation and translocation to the membrane, of sphingosine kinase-1 (SK-1), enzyme that metabolizes sphingosine- 1 phosphate (s1p). Furthermore, FHL-2 siRNA increased significantly (2 fold) stromal derived factor (SDF) -1- induced EPC migration; through upregulation of α-v/β-3 and α-v/β-5 integrins; this was associated with an increase of the F-actin binding protein cortactin, known to promote migration. Interestingly, increased SDF-1- induced EPC migration and upregulation of cortactin by FHL-2 siRNA were totally prevented by CAY10621 , a specific inhibitor of SK-1. In addition stimulation of EPCs with exogenous s1p peptide significantly decreased apoptosis and increased SDF-1- induced migration. These results were confirmed In vivo using FHL-2 knockout (FHL-2 -/-) mice. Moreover, apoptosis was significantly decreased and migration increased in endothelial cells exposed to the conditioned medium of FHL-2 -/- vs. WT EPCs. These effects were abolished by VPC23019 , an antagonist of sphingosine- 1- phosphate receptor- 1 and 3. Finally, reendothelialization after focal carotid endothelial electric injury in WT mice was significantly increased after application of spleen-derived progenitor cells from FHL-2 -/- mice vs. WT mice. Conclusions: Our findings suggest that FHL-2 negatively regulates early outgrowth EPC function and secretion of paracrine factors. FHL-2 inhibition reduces apoptosis, enhances survival and migratory capacity of EPCs and ECs by upregulating SK-1/s1p pathway, integrin subunits and cortactin; which results in the improvement of endothelial regeneration.

Endothelial progenitor cell-based therapy for hemophilia A

As shown by the results of both pre-clinical and clinical studies reported in past decades, the goal of establishing an effective and successful gene therapy for hemophilia A remains feasible and realistic. However, at this time, no single approach has been shown to be clearly superior, and a number of recurring challenges remain to be overcome. Given the persistent problems presented by the host immune response to systemic in vivo gene delivery, and the additional obstacles of inadequate transgene delivery and expression, we propose a re-evaluation of an ex vivo gene transfer approach that utilizes a genetically modified stem cell population. In this strategy, autologous blood outgrowth endothelial progenitor cells are obtained from hemophilic animals, into which a normal copy of the factor VIII gene is introduced via an engineered virus. Cell numbers are expanded in culture prior to their re-implantation under the skin of the hemophilic animals in an artificially developed supporting environment. Follow-up assessment of the treatment involves the general evaluation of clotting activity, the specific measurement of factor VIII levels in the blood, and clinical observation.

Late outgrowth endothelial progenitor cells engineered for improved survival and maintenance of function in transplant-related injury

Chronic allograft vasculopathy (CAV) is a major cause of organ transplant failure that responds poorly to treatment. Endothelial activation, dysfunction and apoptosis contribute to CAV, whereas strategies for protecting endothelium and maximizing endothelial repair may diminish it. Late outgrowth endothelial progenitor cells (LO-EPC) can home to areas of injury and integrate into damaged vessels, implying a role in vascular repair; however, in an allograft, LO-EPC would be exposed to the hazardous microenvironment associated with transplant-related ischaemia reperfusion (I/R) injury and persistent inflammation. We evaluated the in vitro effect of I/R injury and the proinflammatory cytokine tumour necrosis factor (TNF)-α on LO-EPC phenotype and function. We show that LO-EPC are intrinsically more tolerant than mature EC to I/R injury induced apoptosis, maintaining their proliferative, migratory and network formation capacity. Under inflammatory conditions, LO-EPC were activated and released higher levels of inflammatory cytokines, upregulated adhesion molecule expression, and were more susceptible to apoptosis. Lentiviral vector-mediated overexpression of the protective gene A20 in LO-EPC maintained their angiogenic phenotype and function, and protected them against TNF-α-mediated apoptosis, reducing ICAM-1 expression and inflammatory cytokine secretion. Administration of ex vivo modified LO-EPC overexpressing A20 might effect vascular repair of damaged allografts and protect from CAV.

Modulation of chemotactic and pro-inflammatory activities of endothelial progenitor cells by hepatocellular carcinoma

Endothelial progenitor cells (EPCs) participate in the neovascularization processes in the development of hepatocellular carcinoma (HCC). We investigated whether interactions between EPCs and HCC cells affect chemotactic and pro-inflammatory activities of EPCs. Two distinct phenotypes of circulating EPCs, i.e., myeloid-derived EPCs (colony forming unit-endothelial cells, CFU-ECs) and outgrowth EPCs (endothelial-colony forming cells, ECFCs), were co-cultured with Huh7 and Hep3B cells by using transwell chamber and IBIDITM Culture-Inserts and μ-slide plates. Transwell and horizontal migration/invasion assays and time-lapse microscopy were used to monitor and analyze the migration and invasion of EPCs induced by these HCC cells. A human cytokine antibody array was used to compare protein expression profiles in EPCs and HCC cells. Flow cytometry and electromobility shift analysis were used to detect nuclear factor-κB (NF-κB)–DNA binding activity and pro-inflammatory adhesion molecule expression in EPCs. Ectopic full-length CC chemokine receptor 6 (CCR6) plasmid was used to transfect into ECFCs to investigate the role of CCR6 in HCC-induced EPC migration and invasion. The results show that co-culture with Huh7 and Hep3B cells induces the expression of endothelial cell (EC) markers KDR, Flt1, CD31 and VE-cadherin in CFU-ECs, but down-regulates the expressions of CD31 and VE-cadherin in ECFCs. These HCC cells induce migration and invasion of CFU-ECs, but not ECFCs, and do not affect the cell cycle distribution in these EPCs. Cytokine protein array identifies macrophage inflammatory protein-3α (MIP-3α) produced by HCC cells as a critical factor responsible for the HCC-induced chemotaxis of CFU-ECs, which highly express the specific MIP-3α counterreceptor CCR6. Overexpressing CCR6 in ECFCs significantly increases their chemotaxis in response to HCC cells. Co-culturing EPCs with HCC cells results in decreases in NF-κB binding activity and hence intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expressions in EPCs. Our results indicate that HCC cells exert differential effects on CFU-ECs and ECFCs, with increased chemotaxis for CFU-ECs, but not ECFCs. This HCC-induced chemotaxis of CFU-ECs is mediated by MIP-3α produced by HCC cells, which targets to CCR6 on CFU-ECs. Tumors may provide a humoral microenvironment to attenuate the pro-inflammatory activity of EPCs, which might be associated with the tumor escape mechanism.

Statins Enhance Clonal Growth of Late Outgrowth Endothelial Progenitors and Increase Myocardial Capillary Density in the Chronically Ischemic Heart

BackgroundCoronary artery disease and ischemic heart disease are leading causes of heart failure and death. Reduced blood flow to heart tissue leads to decreased heart function and symptoms of heart failure. Therapies to improve heart function in chronic coronary artery disease are important to identify. HMG-CoA reductase inhibitors (statins) are an important therapy for prevention of coronary artery disease, but also have non-cholesterol lowering effects. Our prior work showed that pravastatin improves contractile function in the chronically ischemic heart in pigs. Endothelial progenitor cells are a potential source of new blood vessels in ischemic tissues. While statins are known to increase the number of early outgrowth endothelial progenitor cells, their effects on late outgrowth endothelial progenitor cells (LOEPCs) and capillary density in ischemic heart tissue are not known. We hypothesized that statins exert positive effects on the mobilization and growth of late outgrowth EPCs, and capillary density in ischemic heart tissue.Methodology/Principal FindingsWe determined the effects of statins on the mobilization and growth of late outgrowth endothelial progenitor cells from pigs. We also determined the density of capillaries in myocardial tissue in pigs with chronic myocardial ischemia with or without treatment with pravastatin. Pravastatin therapy resulted in greater than two-fold increase in CD31+ LOEPCs versus untreated animals. Addition of pravastatin or simvastatin to blood mononuclear cells increased the number of LOEPCs greater than three fold in culture. Finally, in animals with chronic myocardial ischemia, pravastatin increased capillary density 46%.ConclusionsStatins promote the derivation, mobilization, and clonal growth of LOEPCs. Pravastatin therapy in vivo increases myocardial capillary density in chronically ischemic myocardium, providing an in vivo correlate for the effects of statins on LOEPC growth in vitro. Our findings provide evidence that statin therapy can increase the density of capillaries in the chronically ischemic heart.

Patient-derived endothelial progenitor cells improve vascular graft patency in a rodent model

Late outgrowth endothelial progenitor cells (EPCs) derived from the peripheral blood of patients with significant coronary artery disease were sodded into the lumens of small diameter expanded polytetrafluoroethylene (ePTFE) vascular grafts. Grafts (1mm inner diameter) were denucleated and sodded either with native EPCs or with EPCs transfected with an adenoviral vector containing the gene for human thrombomodulin (EPC+AdTM). EPC+AdTM was shown to increase the in vitro rate of graft activated protein C (APC) production 4-fold over grafts sodded with untransfected EPCs (p<0.05). Unsodded control and EPC-sodded and EPC+AdTM-sodded grafts were implanted bilaterally into the femoral arteries of athymic rats for 7 or 28days. Unsodded control grafts, both with and without denucleation treatment, each exhibited 7day patency rates of 25%. Unsodded grafts showed extensive thrombosis and were not tested for patency over 28days. In contrast, grafts sodded with untransfected EPCs or EPC+AdTM both had 7day patency rates of 88–89% and 28day patency rates of 75–88%. Intimal hyperplasia was observed near both the proximal and distal anastomoses in all sodded graft conditions but did not appear to be the primary occlusive failure event. This in vivo study suggests autologous EPCs derived from the peripheral blood of patients with coronary artery disease may improve the performance of synthetic vascular grafts, although no differences were observed between untransfected EPCs and TM transfected EPCs.

Use of autologous blood-derived endothelial progenitor cells at point-of-care to protect against implant thrombosis in a large animal model

Titanium (Ti) is commonly utilized in many cardiovascular devices, e.g. as a component of Nitinol stents, intra- and extracorporeal mechanical circulatory assist devices, but is associated with the risk of thromboemboli formation. We propose to solve this problem by lining the Ti blood-contacting surfaces with autologous peripheral blood-derived late outgrowth endothelial progenitor cells (EPCs) after having previously demonstrated that these EPCs adhere to and grow on Ti under physiological shear stresses and functionally adapt to their environment under flow conditions ex vivo. Autologous fluorescently-labeled porcine EPCs were seeded at the point-of-care in the operating room onto Ti tubes for 30 min and implanted into the pro-thrombotic environment of the inferior vena cava of swine ( n = 8). After 3 days, Ti tubes were explanted, disassembled, and the blood-contacting surface was imaged. A blinded analysis found all 4 cell-seeded implants to be free of clot, whereas 4 controls without EPCs were either entirely occluded or partially thrombosed. Pre-labeled EPCs had spread and were present on all 4 cell-seeded implants while no endothelial cells were observed on control implants. These results suggest that late outgrowth autologous EPCs represent a promising source of lining Ti implants to reduce thrombosis in vivo.

Angiotensin II Impairs Endothelial Progenitor Cell Number and Function In Vitro and In Vivo

Endothelial progenitor cells (EPCs) contribute to endothelial regeneration. Angiotensin II (Ang II) through Ang II type 1 receptor (AT(1)-R) activation plays an important role in vascular damage. The effect of Ang II on EPCs and the involved molecular mechanisms are incompletely understood. Stimulation with Ang II decreased the number of cultured human early outgrowth EPCs, which express both AT(1)-R and Ang II type 2 receptor, mediated through AT(1)-R activation and induction of oxidative stress. Ang II redox-dependently induced EPC apoptosis through increased apoptosis signal-regulating kinase 1, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase phosphorylation; decreased Bcl-2 and increased Bax expression; and activation of caspase 3 but had no effect on the low cell proliferation. In addition, Ang II impaired colony-forming and migratory capacities of early outgrowth EPCs. Ang II infusion diminished numbers and functional capacities of EPCs in wild-type (WT) but not AT(1)a-R knockout mice (AT(1)a(-/-)). Reendothelialization after focal carotid endothelial injury was decreased during Ang II infusion. Salvage of reendothelialization by intravenous application of spleen-derived progenitor cells into Ang II-treated WT mice was pronounced with AT(1)a(-/-) cells compared with WT cells, and transfusion of Ang II-pretreated WT cells into WT mice without Ang II infusion was associated with less reendothelialization. Transplantation of AT(1)a(-/-) bone marrow reduced atherosclerosis development in cholesterol-fed apolipoprotein E-deficient mice compared with transplantation of apolipoprotein E-deficient or WT bone marrow. Randomized treatment of patients with stable coronary artery disease with the AT(1)-R blocker telmisartan significantly increased the number of circulating CD34/KDR-positive EPCs. Ang II through AT(1)-R activation, oxidative stress, and redox-sensitive apoptosis signal-regulating kinase 1-dependent proapoptotic pathways impairs EPCs in vitro and in vivo, resulting in diminished vascular regeneration.

Angiotensin II Deteriorates Endothelial Progenitor Cells

See related article, pp 394–403 Approximately 15 years ago it was discovered that the bone marrow and blood harbor stem cells with angiogenic potential.1 Because some of these stem cells express endothelial markers, it is believed that they are endothelial progenitor cells (EPCs) that will contribute to the maintenance of the circulatory system and develop into adult endothelial cells. Many studies followed to unravel the physiological role and the therapeutic application of EPCs. All of these research endeavors did not yet lead to a consensus regarding the identification of true EPCs, and EPC quantification remains subjective.2 To complicate matters even more, it has now been proposed that nonendothelial CD31+ bone marrow cells participate in neovascularization.3 It is, therefore, not only complicated for the investigator to compose a satisfactory study design but also for the reader to evaluate the results. Preferably, such studies investigate circulating, as well as cultured EPCs, and provide proof of their involvement in (patho)physiology through functional studies. Identification of circulating EPCs should be identified by combining stem cell markers with endothelial cell markers, whereas EPCs obtained from cultured blood, bone marrow, or spleen-derived mononuclear cells (MNCs) should be identified on the basis of endothelial markers and variables of angiogenic function.2 It is according to these favorable requirements that, in this issue of Hypertension, Endtmann et al4 show that treatment of adherent, cultured human blood MNCs with angiotensin (Ang) II decreases early outgrowth EPC numbers, colony formation, and migration via Ang II type 1 receptor (AT1R) stimulation and that a 12-day infusion of Ang II reduces the numbers of circulating EPCs …

In VitroFunctional Testing of Endothelial Progenitor Cells That Overexpress Thrombomodulin

This study investigated the augmentation of endothelial progenitor cell (EPC) thromboresistance by using gene therapy to overexpress thrombomodulin (TM), an endothelial cell membrane glycoprotein that has potent anti-coagulant properties. Late outgrowth EPCs were isolated from peripheral blood of patients with documented coronary artery disease and transfected with an adenoviral vector containing human TM. EPC transfection conditions for maximizing TM expression, transfection efficiency, and cell viability were employed. TM-overexpressing EPCs had a fivefold increase in the rate of activated protein C production over native EPCs and EPCs transfected with an adenoviral control vector expressing β-galactosidase (p<0.05). TM upregulation caused a significant threefold reduction in platelet adhesion compared to native EPCs, and a 12-fold reduction compared to collagen I-coated wells. Additionally, the clotting time of TM-transfected EPCs incubated with whole blood was significantly extended by 19% over native cells (p<0.05). These data indicate that TM-overexpression has the potential to improve the antithrombotic performance of patient-derived EPCs for endothelialization applications.

Functional Endothelial Progenitor Cells from Cryopreserved Umbilical Cord Blood

Umbilical cord blood (UCB) is recognized as an enriched source of endothelial progenitor cells (EPCs) with potential therapeutic value. Because cryopreservation is the only reliable method for long-term storage of UCB cells, the clinical application of EPCs depends on our ability to acquire them from cryopreserved samples; however, the feasibility of doing so remains unclear. In this study we demonstrate that EPCs can be isolated from cryopreserved UCB-derived mononuclear cells (MNCs). The number of outgrowth EPC colonies that emerged in culture from cryopreserved samples was similar to that obtained from fresh UCB. Furthermore, EPCs obtained from cryopreserved MNCs were phenotypically and functionally indistinguishable from freshly isolated ones, including the ability to form blood vessels in vivo. Our results eliminate the necessity of performing cell isolation procedures ahead of future clinical needs and suggest that EPCs derived from cryopreserved UCB may be suitable for EPC-related therapies.