Late Outgrowth Endothelial Progenitor Cells Research Articles

Clinically compatible advances in blood-derived endothelial progenitor cell isolation and reprogramming for translational applications

The promise of using induced pluripotent stem cells (iPSCs) for cellular therapies has been hampered by the lack of easily isolatable and well characterized source cells whose genomes have undergone minimal changes during their processing. Blood-derived late-outgrowth endothelial progenitor cells (EPCs) are used for disease modeling and have potential therapeutic uses including cell transplantation and the translation of induced pluripotent stem cell (iPSC) derivatives. However, the current isolation of EPCs has been inconsistent and requires at least 40−80 mL of blood, limiting their wider use. In addition, previous EPC reprogramming methods precluded the translation of EPC-derived iPSCs to the clinic. Here a series of clinically-compatible advances in the isolation and reprogramming of EPCs is presented, including a reduction of blood sampling volumes to 10 mL and use of highly efficient RNA-based reprogramming methods together with autologous human serum, resulting in clinically relevant iPSCs carrying minimal copy number variations (CNVs) compared to their parent line.

Development of in vitro endothelialized drug-eluting stent using human peripheral blood-derived endothelial progenitor cells.

We propose in vitro endothelialization of drug-eluting stents (DES) to overcome late stent thrombosis by directly introducing late-outgrowth human endothelial progenitor cells (EPCs) at the target site utilizing abluminal DES. Isolated EPCs were confirmed as late-outgrowth EPCs by flow cytometric analysis. Abluminally paclitaxel-loaded stents were seeded with different cell concentrations and durations to determine optimal seeding conditions, in both uncrimped and crimped configurations. The seeding yield was determined by evaluating the percent coverage of the stent struts' area. The EPC-seeded DES were exposed to arterial shear stress to evaluate the effect of high shear stress on EPCs. To investigate how much paclitaxel elutes during the seeding procedure, a pharmacokinetic analysis was performed. Finally, to validate the proof of concept, EPC-seeded DES were placed on a fibrin matrix with and without smooth muscle cells (SMCs) and cultured for 3 days under perfusion. The seeding procedure resulted in 47% and 26% coverage of the stent surface in uncrimped and crimped conditions, respectively. After the optimal seeding, almost 99% of drug was still available. When EPC-seeded DES were placed on a fibrin matrix and cultured for 3 days, the EPCs confluently covered the stent surface and spread to the surrounding fibrin gel. When EPC-seeded DES were placed on SMC-containing fibrin layers, cells in contact with the struts died. EPCs can be successfully seeded onto DES without losing drug-eluting capability, and EPCs exhibit sufficient proliferative ability. EPC-seeded DES may combine early re-endothelialization ability with the antirestenotic effectiveness of DES.

MicroRNA-126 protects against vascular injury by promoting homing and maintaining stemness of late outgrowth endothelial progenitor cells

BackgroundEndothelial progenitor cells (EPCs) contribute to reendothelialization and neovascularization and protect against vascular injury and ischemia of various organs. We have previously shown downregulation of microRNA (miR)-126 in EPCs from diabetic patients, which contributes to dysfunction of EPCs including impaired migratory ability. The aims of the present study were to examine (1) in vitro the effects of miR-126 on the homing and stemness of late outgrowth EPCs (LOCs), along with relevant signaling pathways, and (2) in vivo the effects of modulating LOCs by manipulating miR-126 expression on LOC homing and reendothelialization of injured arteries in GK rats (a non-obese diabetes model).MethodsRat bone marrow-derived LOCs were transfected with miR-126 inhibitor or lentiviral vectors expressing miR-126. LOC migration was determined by transwell migration assay. CXCR4 expression was measured by real-time PCR, Western blotting, and confocal microscopy while related signaling pathway proteins were measured by Western Blotting. Stemness gene expression, and gene and protein expression and promoter activity of KLF-8 were also measured. LOCs transfected with lenti-miR-126 or miR-126 inhibitor were injected into GK rats with carotid artery injury, and then vascular reendothelialization and the extent of intimal hyperplasia were examined.ResultsLenti-miR-126 increased while miR-126 inhibitor decreased LOC migration and CXCR4 expression on LOCs. miR-126 positively regulated p-ERK, VEGF, p-Akt, and eNOS protein expression, and inhibitors of these proteins blocked miR-126-induced CXCR4 expression and also reduced LOC migration. Overexpression of miR-126 promoted while inhibition of miR-126 suppressed stemness gene expression in LOCs. miR-126 also inhibited gene and protein expression and promoter activity of KLF-8 while shRNA-mediated knockdown of KLF-8 increased stemness gene expression. Upregulation of stemness gene expression by miR-126 overexpression was completely abrogated by co-transfection of lenti-KLF-8 and lenti-miR-126 into LOCs. In GK rats, transplantation of LOCs overexpressing miR-126 enhanced LOC homing and reendothelialization and decreased intimal hyperplasia of injured arteries.ConclusionOur results indicate that miR-126 protects against vascular injury by promoting CXCR4 expression and LOC homing via ERK/VEGF and Akt/eNOS signaling pathways and maintaining stemness via targeting KLF-8.

TERT assists GDF11 to rejuvenate senescent VEGFR2+/CD133+ cells in elderly patients with myocardial infarction

Growth differentiation factor 11 (GDF11) is a transforming growth factor β superfamily member with a controversial role in rejuvenating old stem cells after acute injury in the elderly population. This study aimed to evaluate the effects of telomerase reverse transcriptase (TERT) on GDF11-mediated rejuvenation of senescent late-outgrowth endothelial progenitor cells (EPCs), defined as VEGFR2+/CD133+ cells, in elderly patients with acute myocardial infarction (AMI). We compared the quantity and capabilities of VEGFR2+/CD133+ cells from old (>60 years), middle-aged (45–60 years), and young (<45 years) AMI patients. The decline in circulating count and survival of VEGFR2+/CD133+ cells with age was accompanied by decrease in their TERT and GDF11 expression levels in patients with AMI. Further, upregulation of TERT could trigger GDF11-mediated rejuvenation of old VEGFR2+/CD133+ cells by renewing their survival and angiogenic abilities through activation of canonical (Smad2/3) and noncanonical (eNOS) signaling pathways. Depletion of GDF11 or TERT caused senescence of young VEGFR2+/CD133+ cells leading to impaired vascular function and angiogenesis in vitro and in vivo, whereas adTERT and rhGDF11 rescued this senescence. TERT cooperates with GDF11 to enhance regenerative capabilities of old VEGFR2+/CD133+ cells. When combined with TERT, GDF11 may represent a potential therapeutic target for the treatment of elderly patients with MI.

ENDOTHELIAL PROGENITOR CELLS ENCAPSULATED IN MATRIX-SUPPLEMENTED MICROGEL IMPROVES CELL RETENTION AND THERAPEUTIC EFFICACY IN PULMONARY ARTERIAL HYPERTENSION

Late outgrowth (L) endothelial progenitor cells (EPC) represent a uniform, highly endothelial-like progenitor cell population; however, their therapeutic benefits in models of pulmonary arterial hypertension (PAH) are limited by poor cell persistence, due to rapid cell loss by apoptosis (anoikis) and redistribution to non-target organs. Temporary single-cell microencapsulation (i.e. cocooning) provides a portable stem cell niche, that can promote cell survival and retention in animal models of organ lung and heart injury. We hypothesize that microencapsulation of L-EPC with an agarose hydrogel supplemented with integrin-binding proteins will increase survival and retention of L-EPCs injected into the jugular vein and result in greater therapeutic benefits compared to non-cocooned cells in a rat monocrotaline (MCT) model of PAH. L-EPCs were encapsulated by vortex-emulsion using various concentrations of agarose, together with fibronectin and fibrinogen, and capsule size and initial cell viability were assessed. Encapsulated and non-encapsulated L-EPCs were transduced with luciferase and administered to SD rats three days after injection of MCT. L-EPCs were tracked in vivo by bioluminescence imaging (BLI) to assess cell persistence and bio-distribution for up to three weeks post cell injection. At end-study, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) were assessed for therapeutic efficacy. The initial BLI signals at 15 minutes after delivery were similar in non-cocooned and cocooned L-EPCs; however, only cocooned cells could be detected by BLI after four and 24 hours (28±12% and 12±8% of baseline signal, respectively; p< 0.0001 and 0.05, n=11). Microencapsulation of L-EPCs led to significant improvement in RVSP three weeks after delivery compared to MCT alone (56±24 vs. 80±7 mmHg, respectively; p<0.05), whereas no improvement in pulmonary hemodynamics was seen with delivery of non-encapsulated cells. These results demonstrate that single-cell cocooning can significantly increase retention of L-EPCs within the lungs. Furthermore, even a modest increase in L-EPC persistence over 24 hours can provide an important therapeutic benefit, not seen with non-encapsulated L-EPCs in the rat MCT model of PAH.

Endothelial Progenitor Cells and Cardiovascular Correlates.

Cardiovascular disease is cited as the underlying cause of death in one out of every three deaths within the United States; this burden on the health care system percolates down to affect patients on an individual level. In part, the problem arises from the low regenerative capacity of cardiovascular system cells, for example, cardiac myocytes, and from oxidative stressors to the human body.Endothelial progenitor cells (EPCs) are a type of stem cell, and various clinical conditions including hypertension and renal failure underlie their dysfunction. EPCs are classified as either early or late outgrowth endothelial progenitor cells depending on the time they appear in circulation and at the site of injury after an inciting event. Their function is paracrine through the release of cytokines, growth factors and chemokines such as interleukin-6 and vascular endothelial growth factor, and they are involved in transdifferentiation into vascular smooth muscle cells and potentially cardiac myocytes. They are beneficial to the modification of cardiovascular cell apoptosis, fibrosis, and contractility. In times of stress, the normal function of endothelial progenitor cells is altered; this creates a maladaptive cycle where stress and failed coping mechanisms enhance each other toward the culmination of cardiovascular disease.The development of the cardiovascular system follows gastrulation in the embryonic period, and the cells that form the system are derived from the mesoderm; being mesoderm, the vascular cells exhibit heterogeneity in their origin and function. The need to understand the molecular and cellular regulatory pathways during development can amalgamate efforts of endothelial cell and cardiovascular system pathophysiology for the advancement of patient cardiovascular reserve and function.

337 - Single-cell matrix-supplemented hydrogel cocooning of endothelial progenitor cells improves retention and therapeutic efficacy in pulmonary arterial hypertension

Background: Late outgrowth (L) endothelial progenitor cells (EPC) represent a uniform, highly endothelial-like progenitor cell population; however, their therapeutic benefits in models of pulmonary arterial hypertension (PAH) are limited by poor cell persistence, due to rapid cell loss by apoptosis (anoikis) and redistribution to non-target organs. Temporary microencapsulation (i.e. cocooning) provides a portable stem cell niche, that can promote cell survival and retention in animal models of organ lung and heart injury.

Synthetic microparticles conjugated with VEGF165 improve the survival of endothelial progenitor cells via microRNA-17 inhibition

Several cell-based therapies are under pre-clinical and clinical evaluation for the treatment of ischemic diseases. Poor survival and vascular engraftment rates of transplanted cells force them to work mainly via time-limited paracrine actions. Although several approaches, including the use of soluble vascular endothelial growth factor (sVEGF)—VEGF165, have been developed in the last 10 years to enhance cell survival, they showed limited efficacy. Here, we report a pro-survival approach based on VEGF-immobilized microparticles (VEGF-MPs). VEGF-MPs prolong VEGFR-2 and Akt phosphorylation in cord blood-derived late outgrowth endothelial progenitor cells (OEPCs). In vivo, OEPC aggregates containing VEGF-MPs show higher survival than those treated with sVEGF. Additionally, VEGF-MPs decrease miR-17 expression in OEPCs, thus increasing the expression of its target genes CDKN1A and ZNF652. The therapeutic effect of OEPCs is improved in vivo by inhibiting miR-17. Overall, our data show an experimental approach to improve therapeutic efficacy of proangiogenic cells for the treatment of ischemic diseases.

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called "early" EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While "early" EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas "early" EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation. Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery.

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called "early" EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While "early" EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas "early" EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation. Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Preconditioned Endothelial Progenitor Cells as Biomarker of Vascular Reparation?

The endothelial progenitor cells (EPCs) have defined as cells positively labeled with both hematopoietic stem cells (CD34) and endothelial cell markers predominantly VEGF receptor-2 (VEGFR2) cumulatively. Therefore, there are at least two types of EPCs labelled as early outgrowth EPCs and late outgrowth EPCs probably distinguished their vascular protective ability. Recent animal and clinical studies have shown that reduced number and weak function of EPCs may not only indicate to higher CV risk, but contribute to the impaired heart and vessels reparation. Interestingly, there are some subpopulations of EPCs especially recruited from peripheral blood cells, which may exhibit very variable pro-angiogenic effect and endothelial repair capacity and they are called “preconditioned” EPCs. The aim of the short commentary is depicted the possibilities to use of measurement of traditionally labeled EPCs as biomarker of cardiovascular risk.

Establishment of an Early Vascular Network Promotes the Formation of Ectopic Bone.

Vascularization is crucial for the induction of bone formation. In this study, we investigated the application of two subtypes of peripheral blood-derived endothelial progenitor cells (EPCs) to stimulate vessel formation in ectopic bone constructs. Early and late outgrowth EPCs (E-EPC and L-EPC, respectively) were characterized for their ability to form network structures in vitro and perfused vessels subcutaneously in mice. Only L-EPCs showed the formation of fully connected networks on Matrigel two-dimensional (2D) angiogenesis assays. The presence of multipotent stromal cells (MSCs) inhibited network formation in 2D assays, but stimulated network formation in three-dimensional plugs. In vivo studies revealed that at 2 weeks, the highest incidence of formed perfused vessels was reached by implanted E-EPC/MSC constructs and this could be attributed to the presence of E-EPCs. L-EPCs displayed a significantly lower frequency of blood vessel formation than E-EPCs and this was accompanied by a lowering of total luminal area densities. Nevertheless, combined E-EPC/L-EPC application somewhat increased the percentage incidence of perfused vessels. After 6 weeks, differences in vascularization were still obvious as all three EPC-based constructs contained higher numbers of perfused vessels than constructs containing MSCs alone. Bone was formed in all constructs at an incidence that coincided with high density of perfused vessels after 2 weeks. Altogether, our findings suggest the differential establishment of vascular networks by E-EPCs and L-EPCs and suggest the importance of early vasculogenesis in ectopic bone formation.

Disrupted Endothelial Cell Layer and Exposed Extracellular Matrix Proteins Promote Capture of Late Outgrowth Endothelial Progenitor Cells.

Late outgrowth endothelial progenitor cells (LO-EPC) possess a high proliferative potential, differentiate into vascular endothelial cells (EC), and form networks, suggesting they play a role in vascular repair. However, due to their scarcity in the circulation there is a requirement for ex vivo expansion before they could provide a practical cell therapy and it is currently unclear if they would home and engraft to an injury site. Using an in vitro flow system we studied LO-EPC under simulated injury conditions including EC activation, ischaemia, disrupted EC integrity, and exposed basement membrane. Perfused LO-EPC adhered to discontinuous EC paracellularly at junctional regions between adjacent cells under shear stress 0.7 dyn/cm2. The interaction was not adhesion molecule-dependent and not enhanced by EC activation. LO-EPC expressed high levels of the VE-Cadherin which may explain these findings. Ischaemia reperfusion injury decreased the interaction with LO-EPC due to cell retraction. LO-EPC interacted with exposed extracellular matrix (ECM) proteins, fibronectin and vitronectin. The interaction was mediated by integrins α5β3, αvβ1, and αvβ3. This study has demonstrated that an injured local environment presents sufficient adhesive signals to capture flow perfused LO-EPC in vitro and that LO-EPC have properties consistent with their potential role in vascular repair.

The regulation of endothelial nitric oxide synthase by extracellular matrix in human late outgrowth endothelial progenitor cells

The regulation of endothelial nitric oxide synthase by extracellular matrix in human late outgrowth endothelial progenitor cells

Intravenous Administration of Endothelial Colony-Forming Cells Overexpressing Integrin β1 Augments Angiogenesis in Ischemic Legs.

When injected directly into ischemic tissue in patients with peripheral artery disease, the reparative capacity of endothelial progenitor cells (EPCs) appears to be limited by their poor survival. We, therefore, attempted to improve the survival of transplanted EPCs through intravenous injection and gene modification. We anticipated that overexpression of integrin β1 will enable injected EPCs to home to ischemic tissue, which abundantly express extracellular matrix proteins, the ligands for integrins. In addition, integrin β1 has an independent angiogenesis-stimulating function. Human endothelial colony-forming cells (ECFCs; late-outgrowth EPCs) were transduced using a lentiviral vector encoding integrin β1 (ITGB1) or enhanced green fluorescent protein (GFP). We then locally or systemically injected phosphate-buffered saline or the genetically modified ECFCs (GFP-ECFCs or ITGB1-ECFCs; 1 × 10(5) cells each) into NOD/Shi-scid, IL-2Rγnull mice whose right femoral arteries had been occluded 24 hours earlier. Upregulation of extracellular matrix proteins, including fibronectin, was apparent in the ischemic legs. Four weeks later, blood perfusion of the ischemic limb was significantly augmented only in the ITGB1-ECFC group. Scanning electron microscopy of vascular casts revealed increases in the perfused blood vessels in the ischemic legs of mice in the ITGB1-ECFC group and significant increases in the density of both capillaries and arterioles. Transplanted ECFC-derived vessels accounted for 28% ± 4.2% of the vessels in the ITGB1-ECFC group, with no cell fusion. Intravenous administration of ECFCs engineered to home to ischemic tissue appears to efficiently mediate therapeutic angiogenesis in a mouse model of peripheral artery disease. Significance: The intravenous administration of endothelial colony-forming cells (ECFCs) genetically modified to overexpress integrin β1 effectively stimulated angiogenesis in ischemic mouse hindlimbs. Transplanted ECFCs were observed in the ischemic leg tissue, even at the chronic stage. Moreover, the cells appeared functional, as evidenced by the improved blood flow. The cell type used (ECFCs), the route of administration (intravenous, not directly injected into the affected area), and the use of ligand-receptor interactions (extracellular matrix and integrins) for homing represent substantial advantages over previously reported cell therapies for the treatment of peripheral artery disease.

Therapeutic effects of late outgrowth endothelial progenitor cells or mesenchymal stem cells derived from human umbilical cord blood on infarct repair

BackgroundThis study sought to systematically investigate the derivation of late outgrowth endothelial progenitor cells (late EPC) and mesenchymal stem cells (MSC) from umbilical cord blood (UCB) and to examine their therapeutic effects on myocardial infarction (MI). MethodsThe expression of angiogenic genes was determined by qRT-PCR. Myocardial infarction (MI) was induced in rats, and cells were directly transplanted into the border regions of ischemic heart tissue. ResultsCulture of UCB mononuclear cells yielded two distinct types of cells by morphology after 2weeks in the same culture conditions. These cells were identified as late EPC and MSC, and each was intramyocardially injected into rat hearts after induction of MI. Echocardiography and histologic analyses demonstrated that both EPC and MSC improved cardiac function and enhanced vascularization, although fibrosis was reduced only in the EPC transplanted hearts. Different paracrine factors were enriched in EPC and MSC. However, once injected into the hearts, they induced similar types of paracrine factors in the heart. Transplanted EPC or MSC were mostly localized at the perivascular areas. This study demonstrated that EPC and MSC can be simultaneously derived from UCB under the same initial culture conditions, and that common paracrine factors are involved in the repair of MI. ConclusionLate EPC and MSC are effective for infarct repair, apparently mediated through common humoral mechanisms.

Diverse contribution of bone marrow-derived late-outgrowth endothelial progenitor cells to vascular repair under pulmonary arterial hypertension and arterial neointimal formation

AimsIt is still controversial whether bone marrow (BM)-derived endothelial progenitor cells (EPCs) can contribute to vascular repair and prevent the progression of vascular diseases. We aimed to characterize BM-derived EPC subpopulations and to evaluate their therapeutic efficacies to repair injured vascular endothelium of systemic and pulmonary arteries. Methods and resultsBM mononuclear cells of Fisher-344 rats were cultured under endothelial cell-conditions. Early EPCs appeared on days 3–6. Late-outgrowth and very late-outgrowth EPCs (LOCs and VLOCs) were defined as cells forming cobblestone colonies on days 9–14 and 17–21, respectively. Among EPC subpopulations, LOCs showed the highest angiogenic capability with enhanced proliferation potential and secretion of proangiogenic proteins. To investigate the therapeutic effects of these EPCs, Fisher-344 rats underwent wire-mediated endovascular injury in femoral artery (FA) and were concurrently injected intraperitoneally with 60mg/kg monocrotaline (MCT). Injured rats were then treated with six injections of one of three EPCs (1×106 per time). After 4weeks, transplanted LOCs, but not early EPCs or VLOCs, significantly attenuated neointimal lesion formation in injured FAs. Some of CD31+ LOCs directly replaced the injured FA endothelium (replacement ratio: 11.7±7.0%). In contrast, any EPC treatment could neither replace MCT-injured endothelium of pulmonary arterioles nor prevent the progression of pulmonary arterial hypertension (PAH). LOCs modified protectively the expression profile of angiogenic and inflammatory genes in injured FAs, but not in MCT-injured lungs. ConclusionBM-derived LOCs can contribute to vascular repair of injured systemic artery; however, even they cannot rescue injured pulmonary vasculature under MCT-induced PAH.

The biphasic effects of oxidized-low density lipoprotein on the vasculogenic function of endothelial progenitor cells.

Late-outgrowth endothelial progenitor cells (EPCs) are stress-resistant and responsible for reparative functions in the cardiovascular system. Oxidized-LDL (oxLDL) plays a critical role in cardiovascular disease pathogenesis. However, it is largely unknown what the impacts of oxLDL are on late-outgrowth EPCs. This study aimed to investigate the concentration-related effects of oxLDL on EPC functions and related angiogenesis, in vitro and in vivo. In this study, early and late-outgrowth EPCs were generated from circulating human mononuclear cells. oxLDL may regulate EPC vasculogenic function via the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). Lower concentrations (5 μg/mL) of oxLDL can potentiate EPC tube formation in vitro and in vivo by activating eNOS mechanisms, which are mediated by p38 MAPK- and SAPK/JNK-related pathways. Higher concentrations of oxLDL (10-50 μg/mL) impaired EPC function via the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathways and consequent inhibition of eNOS activity, which could be reversed by anti-oxidants (diphenylene iodonium and apocynin) and gp91phox siRNA. In conclusion, oxLDL has concentration-dependent biphasic effects on human late-outgrowth EPC tube formation in vitro and in vivo.

Combinatorial approach for improving the outcome of angiogenic therapy in ischemic tissues

Two major populations of endothelial progenitor cells (EPC), namely endothelial colony forming cells (ECFC, or late outgrowth EPC) and circulating angiogenic cells (CAC, or early outgrowth EPC) have been reported to play important roles in vasculogenesis in numerous pathological conditions. However, the poor retention of cells into the ischemic tissue and neovessel fragility are two major flaws that need to be overcome for successful angiogenic therapy. The objective of this study was to explore and exploit the functional properties of EPC populations in order to increase the effectiveness of post-ischemic cell therapy. The results indicate different, still complementary, effects of the two EPC populations on adherence and proliferation of vascular endothelial cells. Matrigel plug assay and mouse hind limb ischemia model showed that concomitant administration of CAC-secreted factors and ECFC resulted in three-fold increase in local cell retention and improved muscle perfusion, vessel maturation and hind limb regeneration, in comparison to either treatment alone. By concluding, factors secreted by CAC co-administered at the time of ECFC transplantation improve tissue regeneration and vascular repair through stabilization of newly-derived blood vessels.

Angiogenic potential of early and late outgrowth endothelial progenitor cells is dependent on the time of emergence

BackgroundRecent studies have suggested that late-outgrowth endothelial progenitor cells (EPCs) derived from human peripheral blood mononuclear cells (hPBMNCs) might have higher angiogenic potential than classically-defined early-outgrowth EPCs (EOCs). However, it still remains unclear which of “so-called” EPC subpopulations defined in a variety of ways has the highest angiogenic potential. Methods and resultsWe classified hPBMNC-derived EPC subpopulations by the time of their emergence in culture. EOCs were defined as attached cells on culture days 3–7. Late-outgrowth EPCs, defined as the cell forming colonies with cobblestone appearance since day 10, were further classified as follows: “moderate”-outgrowth EPCs (MOCs) emerging on days 10–16, “late”-outgrowth EPCs (LOCs) on days 17–23, and “very late”-outgrowth EPCs (VOCs) on days 24–30. Flow cytometry analyses showed the clear differences of hematopoietic/endothelial markers between EOC (CD31+VE-cadherin−CD34−CD14+CD45+) and LOC (CD31+VE-cadherin+CD34+CD14−CD45−). We found that LOCs had the highest proliferation and tube formation capabilities in vitro along with the highest expression of angiogenic genes including KDR and eNOS. To investigate the in vivo therapeutic efficacies, each EPC subpopulation was intravenously transplanted into immunocompromised mice (total 4×105 cells) after unilateral hindlimb ischemia surgery. The LOC-treated mice exhibited significantly-enhanced blood flow recovery (flow ratios of ischemic/non-ischemic leg: 0.99±0.02 [LOC group] versus 0.67±0.07 to 0.78±0.09 [other groups]; P<0.05) and augmented capillary collateral formation in ischemic leg, which were attributable to their direct engraftment into host angiogenic vessels (approximately 10%) and paracrine effects. ConclusionhPBMNC-derived late-outgrowth EPCs emerging on culture days 17–23 are superior to other EPC subpopulations with regard to therapeutic angiogenic potential.