Late Endothelial Progenitor Cells Research Articles

The Role of Angiogenesis Mediators in the Mobilization of Early and Late Endothelial Progenitor Cells from the Bone Marrow in Coronary Heart Disease

Background. A severe form of coronary heart disease (CHD) is ischemic cardiomyopathy (ICMP), the pathogenesis of which has not been fully studied. Disturbances in the mobilization of endothelial progenitor cells (EPC) due to an imbalance of angiogenesis mediators may exacerbate ischemia in ICMP. The aim — to establish the peculiarities of changes in the balance of early and late EPС and subpopulations of VEGFR2+ cells in the blood and bone marrow in relation to the content of angiogenesis mediators and the number of desquamated endothelial cells (DEK) in the blood of patients with CHD, suffering and not suffering from ICMP. Methods. A single-stage, clinical, controlled (case-control) study was conducted from March 2019 to June 2022. 52 patients with CHD who had a history of myocardial infarction were examined: 30 people suffering from ICMP and 22 people not suffering from ICMP, as well as 15 healthy donors. The content of VEGFR2+, VEGFR2+CD34+CD14+ (early EPC), VEGFR2+CD34+CD14– (late EPC), VEGFR2+CD34–CD14+, VEGFR2+CD34–CD14– cells in patients with CHD in the blood (before surgery) and bone marrow (sampling was performed at the beginning of coronary bypass) and in healthy individuals in the blood, as well as (in both groups) the content of CD45–CD146+ DEC in the blood was determined by flow cytometry. The concentration of SDF-1, VEGF-A, MCP-1, GM-CSF, G-CSF in blood plasma was measured by multiplex immunofluorescence analysis. Results. The development of CHD without cardiomyopathy was accompanied by an increase in the content of VEGFR2+CD34+CD14+ and VEGFR2+ cells (0.74 [0.46; 1.23]% and 10.00 [8.20; 11.60]%, respectively, versus 0.19 [0.13; 0.32]%, p 0.001 and 5.40 [4.30; 6.50]%, p = 0.005) and concentrations of SDF-1, MCP-1, GM-CSF (respectively 60.00 [50.00; 81.00] pg/ml; 223.0 [180.0; 297.0] pg/ml; 2.10 [1.45; 3.40] pg/ml versus 30.00 [5.00; 45.00] pg/ml, p = 0.041; 175.1 [140.0; 204.0] pg/ml, p = 0.046; 0.96 [0.46; 1.41] pg/ml, p = 0.038) in the blood relative to the norm. No such changes were observed in patients with ICMP. Regardless of the presence of ICMP, the content of VEGFR2+CD34+CD14–, VEGFR2+CD34–CD14+, VEGFR2+CD34–CD14– cells, VEGF-A, G-CSF in the blood of patients with CHD varied within physiological values, and the number of DEC exceeded the norm (7.26 [5.43; 17.94]×105/l, p = 0.039). The number of VEGFR2+ cells and their immunophenotypes in the bone marrow of patients with ICMP did not differ from the parameters in patients with CHD without cardiomyopathy. Prolonged bleeding from the venopuncture area was registered in one CHD patient without cardiomyopathy. Conclusion. The development of ICMP is associated with the absence of a compensatory response to atherogenesis in the form of increased mobilization of early EPC from the bone marrow due to the absence of a reaction associated with hyperproduction of SDF-1, MCP-1, GM-CSF, which is characteristic of CHD without cardiomyopathy. The content of EPС, VEGFR2+CD34–CD14+ and VEGFR2+CD34–CD14– cells, VEGF-A and G-CSF in the blood in СHD corresponds to physiological values, regardless of the presence of ICMP. The generation of EPC in the bone marrow in ICMP is not impaired.

Adenosine promoted angiogenesis mediated by the release of small extracellular vesicles from human endothelial progenitor cells.

Endothelial progenitor cells (EPCs) are stem cells mainly derived from bone marrow; from where they migrate to repair and regenerate damaged tissues. eEPCs have been classified into two sub-populations, early (eEPC) and late EPCs (lEPC), depending on maturation stages in vitro. In addition, eEPC release endocrine mediators, including small extracellular vesicles (sEVs), which in turn may enhance the eEPC-mediated wound healing properties. Nevertheless, adenosine contributes to angiogenesis by recruiting eEPC at the injury site. However, whether ARs may enhance the secretome of eEPC, including sEVs, is unknown. Therefore, we aimed to investigate whether AR activation increase the release of sEVs in eEPC, which in turn has paracrine effects on recipient endothelial cells. Results shown that 5'-N-ethylcarboxamidoadenosine (NECA), a non-selective agonist, increase both the protein levels of the vascular endothelial growth factor (VEGF), and the number of sEVs released to the conditioned medium (CM) in primary culture of eEPC. Importantly, CM and EVs harvested from NECA-stimulated eEPC promote in vitro angiogenesis, without changes in cell proliferation, in recipient ECV-304 endothelial cells. This constitutes the first evidence showing that adenosine enhances sEVs release from eEPC, which has pro-angiogenic capacity on recipient endothelial cells.

Beyond brain injury biomarkers: chemoattractants and circulating progenitor cells as biomarkers of endogenous rehabilitation effort in preterm neonates with encephalopathy.

Preclinical work and studies in adults have shown that endogenous regeneration efforts that involve mobilization of progenitor cells take place after brain injury. However, kinetics of endogenous circulating progenitor cells (CPCs) in preterm neonates is not well described, particularly their possible role regarding brain injury and regeneration. We aimed to assess the kinetics of CPCs in neonates with encephalopathy of prematurity in relation to brain injury biomarkers, chemoattractants and relevant antenatal and postanal clinical factors, in an effort to outline the related pathophysiology. 47 preterm neonates (of 28-33 weeks GA) were enrolled: 31 newborns with no or minimal brain injury (grade I IVH) and 16 prematures with encephalopathy (grade III or IV IVH, PVL or infarct). Peripheral blood samples obtained on days 1, 3, 9, 18 and 45 after birth were analyzed using flow cytometry, focusing on EPCs (early and late Endothelial Progenitor Cells), HSCs (Hematopoietic Stem Cells) and VSELs (Very Small Embryonic-Like Stem Cells). At the same time-points serum levels of S100B, Neuron-specific Enolase (NSE), Erythropoietin (EPO), Insulin-like growth factor-1 (IGF-1) and SDF-1 were also measured. Neonates were assessed postnatally with brain MRI, and with Bayley III developmental test at 2 years of corrected age. Preterms with brain injury proved to have significant increase of S100B and NSE, followed by increase of EPO and enhanced mobilization mainly of HSCs, eEPCs and lEPCs. IGF-1 was rather decreased in this group of neonates. IGF-1 and most CPCs were intense decreased in cases of antenatal or postnatal inflammation. S100B and NSE correlated with neuroimaging and language scale in Bayley III test, providing good prognostic ability. The observed pattern of CPCs' mobilization and its association with neurotrophic factors following preterm brain injury indicate the existence of an endogenous brain regeneration process. Kinetics of different biomarkers and associations with clinical factors contribute to the understanding of the related pathophysiology and might help to early discriminate neonates with adverse outcome. Timely appropriate enhancement of the endogenous regeneration effort, when it is suppressed and insufficient, using neurotrophic factors and exogenous progenitor cells might be a powerful therapeutic strategy in the future to restore brain damage and improve the neurodevelopmental outcome in premature infants with brain injury.

Decreased T530-pSIRT1 Expression in CD45- Cells After Red Grape Administration

T530-pSIRT1 is one of the biomarkers that shows senescence activity. CD45- cells are the phenotype for late Endothelial Progenitor Cells (EPCs) expressing endothelial antigens. Resveratrol in red grapes is thought to be able to increase T530-pSIRT1 expression and improve endothelial quality. This study aimed to determine the change in T530-pSIRT1 expression by CD45- cells after the administration of red grapes. This study had a quasi-experimental pre-test-post-test one-group design. Research subjects were taken using consecutive sampling methods in the adult population aged 45-55 years. Expression of T530-pSIRT1 was analyzed from the number of CD45- cells and Mean Fluorescence Index (MFI) of CD45- cells using the immune flow cytometry method. Statistical analysis used GraphPad version 9.2.0. The number of research subjects was 17 people with a mean age of 47.3 years and 52.9% were female. There was a significant decrease in the number of CD45- cells (p=0.02) and a significant decrease in T530-pSIRT1 expression indicated by MFI CD45- cells, which were significant (p = < 0.0001). Decrease in T530-pSIRT1 expression in CD45- cells is thought to be caused by several factors that cannot be controlled during the study subject's consumption of red grapes such as diet, exercise, mental stress, and rest periods. Further research is needed to determine the appropriate dose and timing of red grape consumption to increase SIRT1 levels. Consumption of red grapes decreased expression of T530-pSIRT1, which could be caused by the dose and time of consumption of red grapes and the lifestyle of the research subjects that could not be controlled.

CD34+ progenitor cells as diagnostic and therapeutic targets in Alzheimer's disease.

CD34+ progenitor cells as diagnostic and therapeutic targets in Alzheimer's disease.

Exploring Endothelial Colony-Forming Cells to Better Understand the Pathophysiology of Disease: An Updated Review.

Endothelial cell (EC) dysfunction has been implicated in a variety of pathological conditions. The collection of ECs from patients is typically conducted postmortem or through invasive procedures, such as surgery and interventional procedures, hampering efforts to clarify the role of ECs in disease onset and progression. In contrast, endothelial colony-forming cells (ECFCs), also termed late endothelial progenitor cells, late outgrowth endothelial cells, blood outgrowth endothelial cells, or endothelial outgrowth cells, are obtained in a minimally invasive manner, namely, by the culture of human peripheral blood mononuclear cells in endothelial growth medium. ECFCs resemble mature ECs phenotypically, genetically, and functionally, making them excellent surrogates for ECs. Numerous studies have been performed that examined ECFC function in conditions such as coronary artery disease, diabetes mellitus, hereditary hemorrhagic telangiectasia, congenital bicuspid aortic valve disease, pulmonary arterial hypertension, venous thromboembolic disease, and von Willebrand disease. Here, we provide an updated review of studies using ECFCs that were performed to better understand the pathophysiology of disease. We also discuss the potential of ECFCs as disease biomarkers and the standardized methods to culture, quantify, and evaluate ECFCs and suggest the future direction of research in this field.

A Novel Approach to Enhance the Regenerative Potential of Circulating Endothelial Progenitor Cells in Patients with End-Stage Kidney Disease.

Circulating bone marrow-derived endothelial progenitor cells (EPCs) facilitate vascular repair in several organs including the kidney but are progressively diminished in end-stage kidney disease (ESKD) patients, which correlates with cardiovascular outcomes and related mortality. We thus determined if enhancing the tissue-reparative effects of human bone marrow-derived mesenchymal stromal cells (BM-MSCs) with the vasculogenic effects of recombinant human relaxin (RLX) could promote EPC proliferation and function. CD34+ EPCs were isolated from the blood of healthy and ESKD patients, cultured until late EPCs had formed, then stimulated with BM-MSC-derived condition media (CM; 25%), RLX (1 or 10 ng/mL), or both treatments combined. Whilst RLX alone stimulated EPC proliferation, capillary tube formation and wound healing in vitro, these measures were more rapidly and markedly enhanced by the combined effects of BM-MSC-derived CM and RLX in EPCs derived from both healthy and ESKD patients. These findings have important clinical implications, having identified a novel combination therapy that can restore and enhance EPC number and function in ESKD patients.

Exogenous H2S reverses high glucose-induced endothelial progenitor cells dysfunction via regulating autophagy

ABSTRACT This study aims to determine the effect of exogenous hydrogen sulfide (H2S) under high glucose (HG)-induced injury in endothelial progenitor cells (EPCs), and to explore the possible underlying mechanisms. Mononuclear cells were isolated from the peripheral blood of healthy volunteers by density-gradient centrifugation and identified as late EPCs by immunofluorescence and flow cytometry. EPCs were treated with high concentrations of glucose, H2S, Baf-A1, 3-MA or rapamycin. Cell proliferation, cell migration and tube formation were measured using cell counting kit-8, Transwell migration and tube formation assays, respectively. Cellular autophagy flux was detected by RFP-GFP-LC3, and Western blotting was used to examine the protein expression levels of LC3B, P62, and phosphorylated endothelial nitric oxide synthase (eNOS) at Thr495 (p-eNOSThr495). Reactive oxygen species (ROS) levels were measured using a DHE probe. H2S and rapamycin significantly reversed the inhibitory effects of HG on the proliferation, migration, and tube formation of EPCs. Moreover, H2S and rapamycin led to an increase in the number of autophagosomes accompanied by a failure in lysosomal turnover of LC3-II or p62 and p-eNOSThr495 expression and ROS production under the HG condition. However, Baf-A1 and 3-MA reversed the effects of H2S on cell behavior. Collectively, exogenous H2S ameliorated HG-induced EPC dysfunction by promoting autophagic flux and decreasing ROS production by phosphorylating eNOSThr495.

Protocol update for late endothelial progenitor cells identified by double‐positive staining

Endothelial progenitor cells (EPCs), which are precursors of endothelial cells (ECs), have the capacity to circulate, proliferate and differentiate into mature ECs. EPCs are primarily identified by the uptake of 1,1‐dioctadecyl‐3,3,3,3‐tetramethylindocarbocyanine‐labelled acetylated low‐density lipoprotein (Dil‐acLDL) and the binding of fluorescein‐isothiocyanate (FITC)‐conjugated Ulex europaeus agglutinin lectin (FITC‐UEA‐I). However, the cytoplasm and nucleus are usually stained by FITC‐UEA‐I via a typical method to double‐stain late EPCs. It is necessary to explore a new method to improve the quality of fluorescence photomicrographs of late EPCs stained with FITC‐UEA‐I. Here, we described an updated protocol for double‐staining late EPCs with Dil‐acLDL and FITC‐UEA‐I, with the cells more optimally stained with FITC‐UEA‐I.

Up-regulation of GLP-1R improved the dysfunction of late EPCs under hyperglycemia by regulating SIRT1 expression

The dysfunction of endothelial progenitor cells (EPCs) is closely associated with diabetic vascular complications. Both glucagonlike peptide-1 receptor (GLP-1R) and silent information regulator 1 (SIRT1) can control systemic glucose homeostasis and protect endothelial cells against hyperglycemia-induced oxidative stress. In this study, we mainly assessed the role played by SIRT1 and GLP-1R and their relationship in regulating the function of late EPCs under hyperglycemia stimulation. Human peripheral blood mononuclear cells (PBMCs) were cultured in EGM-2 medium and induced to differentiate into EPCs and 25 mM glucose was used to stimulate EPCs to obtain a hyperglycemia condition. Subsequently, the expression and location of GLP-1R and SIRT1 in EPCs were detected. After GLP-1R or SIRT1 knockdown, or the treatment by GLP-1R agonist and/or SIRT1 agonist/inhibitor, the effects of SIRT1 and GLP-1R and their relationship in regulating the function of late EPCs under hyperglycemia stimulation was studied by detecting the apoptosis, migration, adhesion and angiogenicity abilities of EPCs. Results demonstrated that, in high-glucose stimulated EPCs, the expression of GLP-1R and SIRT1 was down-regulated. The knockdown of either GLP-1R or SIRT1 could increase EPCs apoptosis and weaken the migration, adhesion and angiogenicity abilities of EPCs. In addition, the improvement effects of Exendin-4 or GLP-1R over-expression on EPCs dysfunction could be weakened to some degree under SIRT1 knockdown. In conclusion, both GLP-1R and SIRT1 expression played important roles in regulating EPCs dysfunction under hyperglycemia and the up-regulation of GLP-1R improved the dysfunction of late EPCs by regulating SIRT1 expression.

Differences in senescence of late Endothelial Progenitor Cells in non-smokers and smokers.

INTRODUCTIONEndothelial Progenitor Cells (EPCs) are part of hematopoietic stem cells that differentiate into endothelial cells during their blood vessels’ maturation process. The role of EPCs is widely known to contribute to repair of the vascular wall when endothelial dysfunction occurs. However, various risk factors for cardiovascular disease (CVD) influence EPC performance, leading to endothelial dysfunction. One EPC dysfunction is decreased amount of EPC mobilization to the injured tissue. EPC dysfunction reduces the angiogenetic function of EPCs. The vital maturation process that the EPCs must pass is the late phase. The dysfunction of late EPCs is known as senescence. This study aimed to identify and compare senescence of late EPCs, through CD62E and CD41 markers, in non-smokers and smokers as a risk factor for CVD.METHODSEPC collection was from peripheral mononuclear cells (PBMCs) in non-smokers (n=30) and smokers (n=31). The EPCs were then marked by CD62E/CD41 and senescence β-galactosidase assay using FACS. Identification of senescence cells was based on fluorescence with DAPI.RESULTSPositive percentage of late EPCs in non-smokers was not significantly different from that in smokers (p=0.014). The number of senescent late EPCs in smokers was higher than in non-smokers (p<0.0001).CONCLUSIONSEndothelial progenitor cells that experienced senescence in the smokers showed EPC dysfunction, which resulted in decreased cell angiogenic function. Further research is needed to explain the mechanism of re-endothelialization failure in EPC dysfunction due to smoking.

CHIR99021 Augmented the Function of Late Endothelial Progenitor Cells by Preventing Replicative Senescence.

Endothelial progenitor cells (EPCs) are specialized cells in circulating blood, well known for their ability to form new vascular structures. Aging and various ailments such as diabetes, atherosclerosis and cardiovascular disease make EPCs vulnerable to decreasing in number, which affects their migration, proliferation and angiogenesis. Myocardial ischemia is also linked to a reduced number of EPCs and their endothelial functional role, which hinders proper blood circulation to the myocardium. The current study shows that an aminopyrimidine derivative compound (CHIR99021) induces the inhibition of GSK-3β in cultured late EPCs. GSK-3β inhibition subsequently inhibits mTOR by blocking the phosphorylation of TSC2 and lysosomal localization of mTOR. Furthermore, suppression of GSK-3β activity considerably increased lysosomal activation and autophagy. The activation of lysosomes and autophagy by GSK-3β inhibition not only prevented replicative senescence of the late EPCs but also directed their migration, proliferation and angiogenesis. To conclude, our results demonstrate that lysosome activation and autophagy play a crucial role in blocking the replicative senescence of EPCs and in increasing their endothelial function. Thus, the findings provide an insight towards the treatment of ischemia-associated cardiovascular diseases based on the role of late EPCs.

Importance of β2AR elevation for re-endothelialization capacity mediated by late endothelial progenitor cells in hypertensive patients.

Dysfunction of late endothelial progenitor cells (EPCs) has been suggested to be associated with hypertension. β2-Adrenergic receptor (β2AR) is a novel and key target for EPC homing. Here, we proposed that attenuated β2AR signaling contributes to EPCs dysfunction, whereas enhanced β2AR signaling restores EPCs' functions in hypertension. EPCs derived from hypertensive patients exhibited reduced cell number, impaired in vitro migratory and adhesion abilities, and impaired re-endothelialization after transplantation in nude mice with carotid artery injury. β2AR expression of EPCs from hypertensive patients was markedly downregulated, whereas the phosphorylation of the p38 mitogen-activated protein kinase (p38-MAPK) was elevated. The cleaved caspase-3 levels were elevated in EPCs. The overexpression of β2AR in EPCs from hypertensive patients inhibited p38-MAPK signaling, whereas it enhanced in vitro EPC proliferation, migration, and adhesion and in vivo re-endothelialization. The β2AR-mediated effects were attenuated by treating the EPCs with a neutralizing monoclonal antibody against β2AR, which could be partially antagonized by the p38-MAPK inhibitor SB203580. Moreover, shear stress stimulation, a classic nonpharmacological intervention, increased the phosphorylation levels of β2AR and enhanced the in vitro and in vivo functions of EPCs from hypertensive patients. Collectively, the current investigation demonstrated that impaired β2AR/p38-MAPK/caspase-3 signaling at least partially reduced the re-endothelialization capacity of EPCs from hypertensive patients. Restoration of β2AR expression and shear stress treatment could improve their endothelial repair capacity by regulating the p38-MAPK/caspase-3 signaling pathway. The clinical significance of β2AR in endothelium repair still requires further investigation.NEW & NOTEWORTHY Impaired β2-adrenergic receptor (β2AR) expression with an elevation of p38-MAPK/caspase-3 signaling at least partially contributes to the decline of re-endothelialization capacity of late endothelial progenitor cells (EPCs) from hypertensive patients. β2AR gene transfer and shear stress treatment improve the late EPC-mediated enhancement of the re-endothelialization capacity in hypertensive patients through activating β2AR/p38-MAPK/caspase-3 signaling. The present study is the first to reveal the potential molecular mechanism of the impaired endothelium-reparative capacity of late EPCs in hypertension after vascular injury and strongly suggests that β2AR is a novel and crucial therapeutic target for increasing EPC-mediated re-endothelialization capacity in hypertension.

Short term in vitro culture of human endothelial progenitor cells; so called early EPC: Does not belong to true endothelial lineage

Background and Aims: Isolation/characterization of Endothelial Progenitor Cells (EPC) is controversial. Two subtypes have been reported in culture: early and late EPCs. We examined whether the early EPCs are true endothelial cells and have a role in angiogenesis.

Shear stress improves the endothelial progenitor cell function via the CXCR7/ERK pathway axis in the coronary artery disease cases

BackgroundDysfunction in the late Endothelial Progenitor Cells (EPCs) is responsible for endothelial repair in patients with Coronary Artery Disease (CAD), and the shear stress is beneficial for EPCs function. However, the impact of shear stress on the capacity of EPCs in CAD patients has not been elucidated yet. The C-X-C chemokine receptor 7/extracellular signal-regulated kinase (CXCR7)/(ERK) pathways are identified to regulate EPCs function in CAD patients. Here, we hypothesize that shear stress upregulates the CXCR7/ERK pathways, which restore the EPCs function in CAD patients.MethodsThe human Peripheral Blood Mononuclear Cells (PBMCs) were collected from healthy adults and CAD patients and then used for EPCs cultivation. The Lv-siRNA for human CXCR7 was transfected into induced EPCs isolated from the CAD patients. Meanwhile, the EPCs from CAD patients were subjected to shear stress generated by a biomimetic device. Next, the cell viability, migration, tube formation, and apoptosis were detected by CCK-8, Transwell assay, Matrigel, and flow cytometry, respectively. Also, the CXCR7/ERK pathways in human EPCs were analyzed by Western blotting and qRT-PCR.ResultCompared to the EPCs collected from normal adults, the CAD patient-derived EPCs showed reduced in vitro vasculogenic capacity. Also, the level of CXCR7 in CAD patient-derived EPCs was significantly reduced compared to the EPCs of healthy subjects. Meanwhile, the extracellular signal-regulated kinase (ERK), which represents a CXCR7 downstream signaling pathway, had decreased phosphorylation level. The shear stress treatment augmented the CXCR7 expression and also elevated ERK phosphorylation, which is comparable to the up-regulation of CAD patient-derived EPCs function. Further, the small interfering RNA (siRNA)-mediated CXCR7 knockdown diminished the enhanced migration, adhesion, and tube formation capacity of shear stress treated CAD patient-derived EPCs.ConclusionUp-regulation of the CXCR7/ERK pathways by shear stress can be a promising new target in enhancing the vasculogenic ability of CAD patient-derived EPCs.

MiR-150 regulates endothelial progenitor cell differentiation via Akt and promotes thrombus resolution

BackgroundDeep venous thrombosis (DVT) constitutes a major global disease burden. Endothelial progenitor cells (EPCs) have been described in association with recanalization of venous thrombus. Furthermore, emerging evidence suggests microRNAs are involved in this progression. The goal of this study was to investigate the influence of miR-150 on the behavior of EPCs and its potential contribution in venous thrombosis resolution.MethodsWe isolated and cultured EPCs from healthy adults. Next, early EPCs or endothelial colony-forming cells (ECFCs or late EPCs) were transfected with miR-150 agomir and antagomir. Gene expression profiles, proliferation, cytokine secretion, and angiogenic capacity of early EPCs and ECFCs were examined. The effects of miR-150 on c-Myb expression and Akt/FOXO1 signaling were also evaluated. Furthermore, a rat model of venous thrombosis was constructed to determine the in vivo function of EPCs.ResultsOur results showed that miR-150 overexpression in early EPCs significantly promoted differentiation to ECFCs and contributed to proliferation and tube formation. However, suppression of miR-150 in late EPCs inhibited proliferation and tube formation. Moreover, we identified that this progression is regulated by inhibition of c-Myb and activation of the Akt/FOXO1 pathway. Our findings also showed that miR-150 led to the enhanced resolution ability of EPCs in a rat venous thrombosis model.ConclusionsIn this study, we present a novel mechanism of miRNA-mediated regulation of EPCs and Akt activation in thrombus resolution.

Systemic Inflammation, Vascular Function, and Endothelial Progenitor Cells after an Exercise Training Intervention in COPD

BackgroundExercise training is a cornerstone of the treatment of chronic obstructive pulmonary disease (COPD) in all disease stages. Data about the training effects with supplemental oxygen in nonhypoxemic patients remains inconclusive. In this study we set out to investigate the training and oxygen effects on inflammatory markers, vascular function, and endothelial progenitor cells in this population of increased cardiovascular risk. MethodsIn this prospective, randomized, double-blind, crossover study, 29 patients with nonhypoxemic COPD performed combined endurance and strength training 3 times a week while breathing medical air or supplemental oxygen for the first 6-week period, and were then reallocated to the opposite gas for the following 6 weeks. Exercise capacity, inflammatory biomarkers, endothelial function (peripheral arterial tone analysis), and endothelial progenitor cells were assessed. Data were also analyzed for a subgroup with endothelial dysfunction (reactive hyperemia index <1.67). ResultsFollowing 12 weeks of exercise training, patients demonstrated a significant improvement of peak work rate and an associated decrease of blood fibrinogen and leptin. Eosinophils were found significantly reduced after exercise training in patients with endothelial dysfunction. In this subgroup, peripheral arterial tone analysis revealed a significant improvement of reactive hyperemia index. Generally, late endothelial progenitor cells were found significantly reduced after the exercise training intervention. Supplemental oxygen during training positively influenced the effect on exercise capacity without impact on inflammation and endothelial function. ConclusionsThis is the first randomized controlled trial in patients with COPD to show beneficial effects of exercise training not only on exercise capacity, but also on systemic/eosinophilic inflammation and endothelial dysfunction.

Isolation and characterization of endothelial progenitor cells from canine bone marrow

ABSTRACT Endothelial progenitor cells (EPC) are located predominantly in the bone marrow. These cells are useful for treating human vascular diseases; they also are a possible target for restricting blood vessel growth for tumors. Little is known about canine EPC. We investigated a bone marrow EPC isolation method that combines the whole bone marrow culture method and the differential adherent speed method using stillborn canines. MTT proliferation, flow cytometry detection, Dil-ac-LDL uptake, FITC-UEA-1 binding and matrigel assays were used to identify and characterize EPC. We isolated two types of EPC: early EPC and late EPC. We found that isolated cells produced typical colony and cobblestone morphology, and were positive for CD31, CD34, CD133 and VEGFR-2. Significant differences were observed in the intensity of expression between early and late EPC, which suggests their different roles during angiogenesis and vasculogenesis. Both early and late EPC were positive for Dil-ac-LDL and FITC-UEA-1, and displayed tube formation when re-suspended in matrigel, both of which are important functional criteria for identifying EPC. Our method is a novel, effective and efficient way to produce enriched EPC.

Late endothelial progenitor cell-capture stents with CD146 antibody and nanostructure reduce in-stent restenosis and thrombosis

The restoration of damaged endothelium is promising to reduce side effects, including restenosis and thrombosis, in the stent treatment for vascular diseases. Current technologies based on drug delivery for these complications still do not satisfy patients due to invariant recurrence rate. Recently, even if one approach was applied to clinical trial to develop the firstly commercialized stent employing circulating endothelial progenitor cells (EPCs) in blood vessels, it resulted in failure in clinical trial. Based on instruction of the failed case, we designed an advanced EPC-capture stent covered with anti-CD146 antibody (Ab) immobilized silicone nanofilament (SiNf) for the highly efficient and specific capture of not early but late stage of EPCs. In vitro cell capture test demonstrates enhanced capture efficiency and adhesion morphology of late EPCs on the modified substrate. The modified substrates could capture 8 times more late EPCs and even 3 times more mesenchymal stem cells (MSCs) as compared to unmodified one. A porcine model with high similarity to human reproduced in vivo results ideally translated from in vitro cell capture results. As restenosis indicators, lumen area, neointimal rate and stenosis area for modified stents were reduced at the range of 30-60% as compared to those for bare metal stent (BMS). Fibrin score indicating thrombosis was lowered less than half as comparing to that on BMS. These inspiring results are attributed to ~2-fold increased endothelial coverage, determined by immuno-histological staining. Taken together, the CD146 Ab-armed nanofilamentous stent could show great performance in the reduction of thrombosis and restenosis through re-endothelialization due to highly efficient specific cell capture. Statement of SignificanceStents have been developed from simple metal stents to functionalized stents for past decades. However, they have still risks to relapse the occlusion in stented arteries. In this paper, we describe the fabrication and optimization of cell capturing stents to maximize the effective re-endothelialization through the serial coating of silicone nanofilaments and anti-CD146 antibody. The nanofilaments increase the amount of coated antibodies and provide the anchoring points of circulating angiogenic cells for strong focal adhesion. We demonstrate high immobilizing ability of circulating angiogenic cells (endotheliali progenitor cells and mesenchymal stem cells) in vitro under similar shear stress to coronary arteries (15 dyne/cm2). Also, we show accelerating re-endothelialization and the efficient prevention of restenosis in porcine coronary arteries in vivo.

Shear Stress Triggers Angiogenesis of Late Endothelial Progenitor Cells via the PTEN/Akt/GTPCH/BH4 Pathway.

Background Shear stress is an effective modulator of endothelial progenitor cells (EPCs) and has been suggested to play an important role in angiogenesis. The phosphatase and tensin homolog (PTEN)/Akt and guanosine triphosphate cyclohydrolase (GTPCH)/tetrahydrobiopterin (BH4) pathways regulate the function of early EPCs. However, the role of these pathways in the shear stress-induced angiogenesis of late EPCs remains poorly understood. Therefore, we aim to investigate whether shear stress could upregulate the angiogenesis capacity of late EPCs and to further explore the possible underlying mechanisms. Methods Late EPCs were subjected to laminar shear stress (LSS), and their in vitro migration, proliferation, and tube formation capacity were determined. In addition, the in vivo angiogenesis capacity was explored, along with the expression of molecules involved in the PTEN/Akt and GTPCH/BH4 pathways. Results LSS elevated the in vitro activities of late EPCs, which were accompanied by downregulated PTEN expression, accelerated Akt phosphorylation, and GTPCH/BH4 pathway activation (all P < 0.05). Following Akt inhibition, LSS-induced upregulated GTPCH expression, BH4, and NO level of EPCs were suppressed. LSS significantly improved the migration, proliferation, and tube formation ability (15 dyn/cm2 LSS vs. stationary: 72.2 ± 5.5 vs. 47.3 ± 7.3, 0.517 ± 0.05 vs. 0.367 ± 0.038, and 1.664 ± 0.315 vs. 1 ± 0, respectively; all P < 0.05) along with the in vivo angiogenesis capacity of late EPCs, contributing to the recovery of limb ischemia. These effects were also blocked by Akt inhibition or GTPCH knockdown (P < 0.05, respectively). Conclusions This study provides the first evidence that shear stress triggers angiogenesis in late EPCs via the PTEN/Akt/GTPCH/BH4 pathway, providing a potential nonpharmacologic therapeutic strategy for promoting angiogenesis in ischemia-related diseases.