Phenotype Of Endothelial Progenitor Cells Research Articles

Age-Related Choroidal Involution Is Associated with the Senescence of Endothelial Progenitor Cells in the Choroid.

Background: Choroidal involution is a common feature of age-related ischemic retinopathies such as age-related macular degeneration (AMD). It is now well recognized that endothelial progenitor cells (EPCs) are essential to endothelial repair processes and in maintaining vascular integrity. However, the contribution of EPCs and the role of senescence in age-related choroidal vascular degeneration remain to be investigated. In this study, we compared the senescent phenotype of EPCs in the choroid and performed whole-genome profiling of EPCs derived from young versus old rats. Methods and Results: We isolated and compared the retinas of young (6-weeks-old) and old (16-18-month-old) rats. The thickness of the choroid and outer nuclear layer (ONL), along with local quantification of CD34+ EPCs, was performed. Compared to young rats, older rats displayed a significant reduction in choroidal and ONL thickness associated with markedly fewer choroid-localized EPCs; this was attested by lower expression of several EPC markers (CXCR4, CD34, CD117, CD133, and KLF-2). Choroid and choroid-localized EPCs displayed abundant senescence as revealed by increased β-gal and P53 expression and decreased Lamin-B1 (immunostaining and RT-qPCR). Concordantly, choroidal cells and EPCs isolated from older rats were unable to form vascular networks ex vivo. To better understand the potential mechanisms associated with the dysfunctional EPCs linked to age-related choroidal involution, we performed whole-genome profiling (mRNA and miRNA) of EPCs derived from old and young rats using next-generation sequencing (NGS); 802 genes were significantly modulated in old vs. young EPCs, corresponding to ~2% of total genes expressed. Using a bioinformatic algorithm, the KEGG pathways suggested that these genes participate in the modulation of several key signaling processes including inflammation, G protein-coupled receptors, and hematopoietic cell lineages. Moreover, we identified 13 miRNAs involved in the regulation of immune system processes, cell cycle arrest and senescence, which are significantly modulated in EPCs from old rats compared to young ones. Conclusions: Our results suggest that age-related choroidal involution is associated with fewer EPCs, albeit displaying a senescence-like phenotype. One would be tempted to propose that biological modification of native EPCs (such as with senolytic agents) could potentially provide a new strategy to preserve the vascular integrity of the aged choroid, and evade progression to degenerative maculopathies.

Circulating levels of endothelial progenitor cells are associated with better cognitive function in older adults with glucagon-like peptide 1 receptor agonist-treated type 2 diabetes

AimsTo evaluate cognitive function in subjects with type 2 diabetes (T2D) treated with glucagon-like peptide 1 receptor agonist (GLP-1RA) plus metformin or metformin alone and its association with endothelial progenitor cells (EPCs). MethodsAdults with T2D treated with GLP-1RA plus metformin (GLP-1RA + MET) or MET alone for at least 12 months were included. Montreal Cognitive Assessment test (MoCA), Mini-Mental State Examination (MMSE), Mini Nutritional Assessment (MNA) and disability tests were administered. Circulating levels of seven EPCs phenotypes were measured by flow cytometry. ResultsA total of 154 elderly patients were included, of whom 78 in GLP-1RA + MET group and 76 in MET group. The GLP-1RA + MET group showed better cognitive function as indicated by a significant higher MoCA and MMSE scores, and higher levels of CD34+ CD133+, CD133+ KDR+, and CD34+ CD133+ KDR+ as compared with MET group. The number of CD34+ CD133+ KDR+ cells was an independent predictor of higher MoCA, MMSE and MNA scores. ConclusionsPeople with T2D on GLP-1RA + MET treatment had better cognitive function and higher circulating levels of EPCs as compared with those on MET alone warranting further studies to understand the interrelationship between EPCs, GLP-RA treatment and cognitive health.

Abstract 15273: Impact of Lipid Lowering Therapy on Progenitor Endothelial Cell Phenotype and Function in Coronary Artery Disease

Introduction: Early revascularization of infarcted zone is crucial to improve patient′s outcomes. Despite their primary actions, major lipid-lowering drugs may also reduce cardiovascular events by pleiotropic mechanisms. Hypothesis: Conventional (statin) and novel lipid-lowering therapies [(PSCK9-inhibitor (PCSK9i) and eicosatetraenoic acid (EPA)] can improve endothelial progenitor cells (EPC) phenotype and favor neovascularization. Methods: EPC were isolated from peripheral blood of patients with acute coronary syndrome (n=62) who were statin-naïve (n=34) or chronic statin treatment (n=28) at time of inclusion. A set of statin-naïve EPC and mature endothelial cells (HUVEC) were treated in vitro with statin (20nM), PCSKi (1μM) and EPA (3μM). EPC phenotype was determined as EPC colony number and time of appearance, proliferative rate, and ability to form capillary-like structures. Serum PCSK9 and markers of angiogenesis and endothelial function were determined by multiplexed protein assay. Results: Frequency of EPC appearance was higher in samples of statin group (78%) compared to naïve group (66%) (Fig A). They also appeared much earlier (Fig B), in greater quantity (4.7 vs 2.1 colony, p = 0.0009) and had higher proliferative rate than EPCs of naïve group. Besides they had increased potential of tube formation and shaped more branched vessel-like structures (Fig C). No differences in serum levels of biomarkers were found. In vitro treatment of EPC with statin, PCSK9i and EPA did not modify colonies behavior (Fig D). PCSK9 levels showed negative correlation with EPC proliferation (r -0.4150, p=0.004), and PCSK9i treatment increased angiogenic potential of HUVEC (Fig E). Conclusions: Statin and PCSK9i have pleiotropic effects on EPC, boosting their potential to form endothelial cells colonies and to induce angiogenesis. These effects could contribute to the progression and outcomes of coronary artery disease. Funding: ISCIII - FEDER-ERDF (PI19/00264)

Upregulated miR-206 Aggravates Deep Vein Thrombosis by Regulating GJA1-Mediated Autophagy of Endothelial Progenitor Cells.

Background Deep vein thrombosis (DVT) is the third most prevalent vascular disease worldwide. MicroRNAs (miRNAs) play regulatory roles in functions of endothelial progenitor cells (EPCs), which is becoming a promising therapeutic choice for thrombus resolution. Nevertheless, the role of miR-206 in EPCs is unclear. Methods EPCs were isolated from the peripheral blood of patients with DVT. In DVT mouse models, DVT was induced by stenosis of the inferior vena cava (IVC). The levels of miR-206 and gap junction protein alpha 1 (GJA1) in EPCs and vascular tissues of DVT mice were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The proliferation, migration, apoptosis, and angiogenesis were tested by cell counting kit-8 (CCK-8) assay, Transwell assay, flow cytometry analysis, and in vitro tube formation assay. The levels of autophagy-related proteins as well as the level of GJA1 in EPCs and vascular tissues were evaluated by western blotting. DVT formation in vivo was observed through hematoxylin-eosin (HE) staining. The expression of thrombus resolution markers, CD34 molecule (CD34) and matrix metallopeptidase 2 (MMP2), in the thrombi was measured by immunofluorescence staining. Results miR-206 overexpression inhibited proliferation, migration, and angiogenesis and promoted apoptosis of EPCs, while miR-206 knockdown exerted an opposite effect on EPC phenotypes. Downregulation of GJA1, the target of miR-206, abolished the influence of miR-206 on EPC phenotypes. Furthermore, silencing of miR-206 suppressed the autophagy of EPCs via upregulating GJA1. miR-206 knockdown repressed thrombus formation, enhanced the homing ability of EPCs to the thrombosis site, and facilitated thrombus resolution in DVT mouse models. Additionally, miR-206 was upregulated while GJA1 was downregulated in vascular tissues of DVT mice. miR-206 knockdown elevated GJA1 expression in vascular tissues of DVT mice. The expression of miR-206 was negatively correlated with that of GJA1 in DVT mice. Conclusion miR-206 knockdown upregulates GJA1 to inhibit autophagy of EPCs and then promote EPC proliferation, migration, and angiogenesis, thereby enhancing EPC homing to thrombi and facilitating thrombus resolution.

Type 1 Diabetes Patients With Different Residual Beta-Cell Function but Similar Age, HBA1c, and Cardiorespiratory Fitness Have Differing Exercise-Induced Angiogenic Cell Mobilisation.

BackgroundMany individuals with type 1 diabetes retain residual beta-cell function. Sustained endogenous insulin and C-peptide secretion is associated with reduced diabetes related complications, but underlying mechanisms remain unclear. Lower circulating numbers of endothelial and hematopoietic progenitor cells (EPCs and HPCs), and the inability to increase the count of these cells in response to exercise, are also associated with increased diabetes complications and cardiovascular disease. It is unknown whether residual beta-cell function influences HPCs and EPCs. Thus, this study examined the influence of residual beta-cell function in type 1 diabetes upon exercise-induced changes in haematopoietic (HPCs) and endothelial progenitor cells (EPCs).MethodsParticipants with undetectable stimulated C-peptide (n=11; Cpepund), 10 high C-peptide (Cpephigh; >200 pmol/L), and 11 non-diabetes controls took part in this observational exercise study, completing 45 minutes of intensive walking at 60% . Clinically significant HPCs (CD34+) and EPCs (CD34+VEGFR2+) phenotypes for predicting future adverse cardiovascular outcomes, and subsequent cell surface expression of chemokine receptor 4 (CXCR4) and 7 (CXCR7), were enumerated at rest and immediately post-exercise by flow cytometry.ResultsExercise increased HPCs and EPCs phenotypes similarly in the Cpephigh and control groups (+34-121% across phenotypes, p<0.04); but Cpepund group did not significantly increase from rest, even after controlling for diabetes duration. Strikingly, the post-exercise Cpepund counts were still lower than Cpephigh at rest.ConclusionsResidual beta-cell function is associated with an intact exercise-induced HPCs and EPCs mobilisation. As key characteristics (age, fitness, HbA1c) were similar between groups, the mechanisms underpinning the absent mobilisation within those with negative C-peptide, and the vascular implications, require further investigation.

Distinctive Roles of YAP and TAZ in Human Endothelial Progenitor Cells Growth and Functions.

The hippo signaling pathway plays an essential role in controlling organ size and balancing tissue homeostasis. Its two main effectors, yes-associated protein (YAP) and WW domain-containing transcription regulator 1, WWTR1 or TAZ, have also been shown to regulate endothelial cell functions and angiogenesis. In this study, the functions of YAP and TAZ in human endothelial progenitor cells (EPCs) were investigated by a loss-of-function study using CRISPR/Cas9-mediated gene knockdown (KD). Depletion of either YAP or TAZ reduced EPC survival and impaired many of their critical functions, including migration, invasion, vessel-formation, and expression of pro-angiogenic genes. Notably, TAZ-KD EPCs exhibited more severe phenotypes in comparison to YAP-KD EPCs. Moreover, the conditioned medium derived from TAZ-KD EPCs reduced the survivability of human lung cancer cells and increased their sensitivity to chemotherapeutic agents. The overexpression of either wild-type or constitutively active TAZ rescued the impaired phenotypes of TAZ-KD EPCs and restored the expression of pro-angiogenic genes in those EPCs. In summary, we demonstrate the crucial role of Hippo signaling components, YAP and TAZ, in controlling several aspects of EPC functions that can potentially be used as a drug target to enhance EPC functions in patients.

Alterations in the Levels of Circulating and Endothelial Progenitor Cells Levels in Young Adults with Type 1 Diabetes: A 2-Year Follow-Up from the Observational METRO Study.

PurposeType 1 diabetes is associated with high risk of cardiovascular disease (CVD). Reduced levels of circulating progenitor cells (CPCs) and endothelial progenitor cells (EPCs) have been indicated as a risk factor for adverse cardiovascular outcomes and death in people at high cardiovascular risk. The aim of the present study was to evaluate the change in CPCs and EPCs levels in a population of young type 1 diabetic patients treated with intensive insulin regimen over a period of 2 years.Patients and MethodsA total of 204 type 1 diabetic patients, of whom 84 treated with insulin pump (CSII) and 120 with multiple daily insulin injections (MDI), completed a 2-year follow-up. Clinical measurements, including the indices of glycemic control and glucose variability, were collected at baseline and after 2 years. Both CPC and EPC cell count were assessed by flow cytometry.ResultsMean age of participants was 24.5 years and mean diabetes duration was 13.6 years. After 2 years, we found a significant reduction of HbA1c (−0.3% versus baseline, P <0.001), associated with decrease in mean amplitude of glucose excursion (MAGE) (−0.5 mmol/L versus baseline, P<0.001), continuous overall net glycemic action (CONGA) (−0.2 mmol/L versus baseline, P=0.006), and blood glucose standard deviation (BGSD) (−0.2 mmol/L versus baseline, P<0.001). The number of all EPCs phenotypes, but not CPC cell count, significantly raised up in the entire population, with higher increase in CSII group. MAGE resulted as an independent predictor for increased levels of both CD34+ (P = 0.020) and CD34+KDR+ (P = 0.004) cell count in the whole population.ConclusionOver a 2-year follow-up, young type 1 diabetic patients showed an increase in circulating EPCs levels, which was higher in patients with CSII. Glucose variability resulted as an independent predictor of the raised levels of EPCs in this selected population.

Endothelial progenitor cells and peripheral neuropathy in subjects with type 2 diabetes mellitus

AimsTo examine for differences in circulating progenitor cells (CPCs) and endothelial progenitor cells (EPCs) in patients with and without diabetic peripheral neuropathy (DPN). MethodsA total of 105 participants were included: 50 patients with type 2 diabetes (T2DM) and DPN, 30 patients with T2DM without DPN and 25 healthy individuals. CPCs and 6 different EPCs phenotypes were assessed with flow cytometry. We also measured plasma levels of vascular endothelial growth factor (VEGF), stromal cell-derived factor 1 (SDF-1), vascular cell adhesion protein-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM) and tumor necrosis factor a (TNFa). ResultsNo difference was observed in the number of CPCs among the 3 groups. Patients with DPN had higher numbers of all 6 EPCs phenotypes when compared with patients without DPN and higher number of 5 EPCs phenotypes when compared with healthy individuals. Plasma VEFG, VCAM-1, ICAM-1 and TNFa levels did not differ among the 3 groups. Patients with DPN had lower SDF-1 levels in comparison with healthy individuals. ConclusionCirculating EPCs are increased while SDF-1 levels are decreased in the presence of DPN. Our findings suggest that DPN may be associated with impaired trafficking of EPCs and impaired EPCs homing to the injured endothelium.

Integrins α4β1 and αVβ3 are Reduced in Endothelial Progenitor Cells from Diabetic Dyslipidemic Mice and May Represent New Targets for Therapy in Aortic Valve Disease

Diabetes reduces the number and induces dysfunction in circulating endothelial progenitor cells (EPCs) by mechanisms that are still uncovered. This study aims to evaluate the number, viability, phenotype, and function of EPCs in dyslipidemic mice with early diabetes mellitus and EPC infiltration in the aortic valve in order to identify possible therapeutic targets in diabetes-associated cardiovascular disease. A streptozotocin-induced diabetic apolipoprotein E knock-out (ApoE−/−) mouse model was used to identify the early and progressive changes, at 4 or 7 days on atherogenic diet after the last streptozotocin or citrate buffer injection. Blood and aortic valves from diabetic or nondiabetic ApoE−/− animals were collected. EPCs were identified as CD34 and vascular endothelial growth factor receptor 2 positive monocytes, and the expression levels of α4β1, αVβ3, αVβ5, β1, αLβ2, α5 integrins, and C-X-C chemokine receptor type 4 chemokine receptor on EPC surface were assessed by flow cytometry. The number of CD34 positive cells in the aortic valve, previously found to be recruited progenitor cells, was measured by fluorescence microscopy. Our results show that aortic valves from mice fed 7 days with atherogenic diet presented a significantly higher number of CD34 positive cells compared with mice fed only 4 days with the same diet, and diabetes reversed this finding. We also show a reduction of circulatory EPC numbers in diabetic mice caused by cell senescence and lower mobilization. Dyslipidemia induced EPC death through apoptosis regardless of the presence of diabetes, as shown by the higher percent of propidium iodide positive cells and higher cleaved caspase-3 levels. EPCs from diabetic mice expressed α4β1 and αVβ3 integrins at a lower level, while the rest of the integrins tested were unaffected by diabetes or diet. In conclusion, reduced EPC number and expression of α4β1 and αVβ3 integrins on EPCs at 4 and 7 days after diabetes induction in atherosclerosis-prone mice have resulted in lower recruitment of EPCs in the aortic valve.

Mechanisms of regeneration of the endothelium at diabetes mellitus

Vascular endothelium is the main organ, suffering from diabetes mellitus (DM) and cardiovascular diseases (CVD). In this review, the role of endothelial progenitor cells (EPCs) in the regeneration of the endothelium and in the formation of new blood vessels is considered. Mechanisms of migration and mobilization of EPCs from the bone marrow to the damage zone are described. The analyzed data show that CVD and DM cause the decrease in the number and the disturbance of the function of EPCs. The data on the heterogeneity of the population of EPCs are presented in article. The various combinations of surface markers for identification of these cells are assessed. At the same time, protocols for the identification of EPCs have not been developed, which confirms the relevance of the search for the phenotype of EPCs, which would be adopted as the standard.

Tumor-endothelial cell interaction in an experimental model of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a densely vascularized tumor that is highly dependent on angiogenic pathways to direct arterial blood flow to the growing neoplasm, though little is known about how the interaction of tumor and endothelial cells drives these processes and the degree of clinical importance. To this end, we examined the intercellular cross-talk between HepG2 (human HCC) and human endothelial progenitor cells (EPC) in a co-culture system that mimics some aspects of initial tumor parenchyma and stroma interactions. The results showed that the remote cell-to-cell (paracrine) interactions between HepG2 cells and EPC play a critical role in the differentiation and angiogenic activity of endothelial cells, possibly through intercellular signaling function of the exosomes released in the medium by HepG2 cells. The tumor-cell activated phenotype of EPC was associated with increased migration and elevated expression of ephrin-B2, and Delta-like 4 ligand (DLL4). Furthermore, ephrin-B2 was found to be overexpressed in HCC and cholangiocarcinoma tissue samples taken from humans. Overall, our results demonstrate that ephrin-B2 and Dll4 mediated co-dependence of HCC and EPC intercellular crosstalk in the initial stages of HCC establishment and development, a promising target for new clinical strategies.

Association of growth-differentiation factor-15 with the number of circulating proangiogenic endothelial progenitor cells in patients with type 2 diabetes mellitus

The objective: to investigate the relationship between levels of growth differentiation factor-15 (GDF-15) and circulating number of endothelial progenitor cells (EPCs) with angiopoietin phenotypes: CD34+CD14+CD309+, and CD34+CD14+CD309+Tie2+ in patients with type 2 DM.&#x0D; Methods: The study was retrospectively involved 76 patients with type 2 DM aged 38 to 55 years and 30 healthy volunteers. Data collection included demographic and anthropometric information, hemodynamic performances and biomarkers of the diseases. Flow cytometry was used to determine EPCs' populations.&#x0D; Results: The levels of GDF-15 in peripheral blood of diabetics associated with age (r = 0.31, P = 0.044), high-sensitive C-reactive protein [hs-CRP] (r = 0.40, P = 0.001), smoking (r = 0.38, P = 0.001), body mass index [BMI] (r = 0.34, P = 0.001), LDL cholesterol (r = 0.28, P = 0.001), glycated hemoglobin [HbA1c] (r = -0.28, P = 0.001), number of CV risk factors (r = 0.26, P = 0.001). In univariate logistic regression analysis we found that level of GDF-15 ≥ 618 pg/mL, hs-CRP ≥7.12 mg/L, HbA1c ≥6.4%, fasting glucose ≥6.7 mmol/L, and BMI ≥27.3 kg/m2 predicted deficiency of both angiopoetic phenotypes of EPCs. In multivariate logistic regression model GDF-15 ≥618 pg/mL demonstrated the best odds ratio values for declining of EPCs in diabetics in comparison with other predictors including BMI, HbA1c and hs-CRP.&#x0D; Conclusion: GDF-15 was remarkably evaluated in type 2 DM population to healthy volunteers, and it was an independent factor that contributes to mobilization and probably proliferation of endothelial precursors with high angiopoetic activity.

Circulating Endothelial Progenitor Cells Present an Inflammatory Phenotype and Function in Patients With Alcoholic Liver Cirrhosis.

Background and Aim: Endothelial progenitor cells (EPCs) have been implicated in liver injury and repair. However, the phenotype and potential of these heterogenous EPCs remain elusive. In particular, their involvement in the pathogenesis of alcoholic liver cirrhosis (ALC) remains unclear. The current study extensively characterized the phenotype and functions of EPCs to understand their role in ALC pathogenesis.Methods: Circulating EPCs were identified as CD34+CD133+CD31+ cells by flow cytometer in ALC patients (n = 7) and healthy controls (HC, n = 7). A comprehensive characterization of circulating EPCs using more than 30 phenotype markers was performed by mass cytometer time of flight (CyTOF) in an independent cohort of age and gender matched ALC patients (n = 4) and controls (n = 5). Ex vivo cultures of circulating EPCs from ALC patients (n = 20) and controls (n = 18) were also tested for their functions, including colony formation, LDL uptake, lectin binding and cytokine secretion (ELISA).Results: Three distinct populations of circulating EPCs (CD34+CD133+CD31+) were identified, classified on their CD45 expression (negative: CD45−; intermediate: CD45int; high: CD45hi). CD45int and CD45hi EPCs significantly increased in ALC patients compared to controls (p-val = 0.006). CyTOF data showed that CD45hi EPCs were distinct from CD45− and CD45int EPCs, with higher expression of T cell and myeloid markers, including CD3, CD4, HLA-DR, and chemokine receptors, CCR2, CCR5, CCR7, and CX3CR1. Similar to circulating EPCs, percentage of CD45hiCD34+CD31+ EPCs in ex-vivo cultures from patients, were significantly higher compared to controls (p < 0.05). Cultured EPCs from patients also showed increased LDL uptake, lectin binding and release of TNF-alpha, RANTES, FGF-2, and VEGF.Conclusions: We report the first extensive characterization of circulating human EPCs with distinct EPC subtypes. Increase in CD45hi EPC subtype in ALC patients with enhanced functions, inflammatory cytokines and angiogenic mediators in patients suggests an inflammatory role for these cells in ALC.

Gender-differences in glycemic control and diabetes related factors in young adults with type 1 diabetes: results from the METRO study.

To describe gender differences concerning glycemic control, cardiovascular risk factors, diabetic complications, concomitant pathologies, and circulating endothelial progenitor cells (EPCs), in a population of young adults with type 1 diabetes. We collected data from 300 consecutively patients (168 males and 132 females), aged 18-30 years, among those admitted at Diabetes Unit of University of Campania "Luigi Vanvitelli" (Naples, Italy) from March 2012 to January 2017. Circulating levels of seven EPCs phenotypes were determined by flow cytometry. As compared to men, women with type 1 diabetes had a significantly higher HbA1c levels (%, 8.4 ± 1.3 vs. 8.1 ± 1.3, P = 0.020), body mass index (Kg/m2, 24.8 ± 4.2 vs. 23.9 ± 3.9, P = 0.034), HDL-cholesterol (mg/dL, 61.7 ± 13.7 vs. 54.7 ± 13.9, P < 0.001), and a lower count of both CD133+KDR+ and CD34+KDR+CD133+ EPCs (P = 0.022, P < 0.001, respectively). A higher proportion of women had overweight/obesity, and thyroiditis; smoking and sexual dysfunctions were more prevalent in men than in women. Young adults with type 1 diabetes present gender differences with regard to glycemic control, prevalence of some cardiovascular risk factors, sexual dysfunctions and circulating levels of EPCs, most often to the detriment of women.

STEM/PROGENITOR CELLS ADHESION STRENGTHENING TO SYNTHETIC MATERIAL USING EXTRACELLULAR MATRIX

Background: Tissue-engineered vascular grafts of small diameter have being widely used in coronary artery bypass grafting. However, the rate of settling of these grafts with endothelial cells is insufficient. Aims:The aim of the study was to selucidate the adhesion of endothelial progenitor cells and mesenchymal stem cells to synthetic materials (polystyrol, polytetrafluoroethylene) preprocessed with extracellular matrix (gelatin, fibronectin, collagen) in vitro.Materials and methods: For the study,the endothelial progenitor cells (EPC) were isolated from the peripheral blood of patients with ischemic heart disease, and mesenchymal stem cells (MSC) — from bone marrow of Wistar rats. Commercial monoclonal antibodies for flow- cytometry were used to determine the phenotype of EPC. To isolate the early and late EPC from mononuclear cells of peripheral blood, cells were raised for 8 and 16 days on a gelatin or fibronectin based substrate. The commercial kits for enzyme linked immunoassay were applied to assess levels of cytokine production and nitric oxide by early and late EPC conditioned by gelatin or fibronectin on the 8th and 16th days of growth. To conduct the study the MSC were isolated from bone marrow of rats. To determine the attachment of adherent fraction of nucleated bone marrow cells, cell morphology, the adipogenic and osteogenic differentiation were evaluated. To assess migration of MSC in real time within 24 hours on the device Cell-IQ, we used «closure/wound healing» test. The commercial kits for enzyme linked immunoassay were applied to assess levels of cytokine production and nitric oxide by MSC; and components of the extracellular matrix (fibronectin, collagen I and type IV) ― to assess the increased adhesion of MSC to the polytetrafluoroethylene. Results: The results demonstrated that endothelial progenitor cells adhere to both gelatin and fibronectin and confirmed the influence of these extracellular matrix components on the cytokine levels produced by early and late endothelial cells. The combination of fibronectin with type I or IV collagen or the combination of thereof promotes the adhesion to polytetrafluoroethylene and colonization of the graft.Conclusions: Preprocessing of synthetic material (polystyrene, polytetrafluoroethylene) enhances adhesion and growth of EPC and MSC which can be implemented when creating tissue-engineered vascular grafts for small diameter coronary artery bypass grafting with specified conditions of settlement by the cells involved in neointima formation.

TRPC3-mediated Ca2+ signals as a promising strategy to boost therapeutic angiogenesis in failing hearts: The role of autologous endothelial colony forming cells.

Endothelial progenitor cells (EPCs) are a sub-population of bone marrow-derived mononuclear cells that are released in circulation to restore damaged endothelium during its physiological turnover or rescue blood perfusion after an ischemic insult. Additionally, they may be mobilized from perivascular niches located within larger arteries' wall in response to hypoxic conditions. For this reason, EPCs have been regarded as an effective tool to promote revascularization and functional recovery of ischemic hearts, but clinical application failed to exploit the full potential of patients-derived cells. Indeed, the frequency and biological activity of EPCs are compromised in aging individuals or in subjects suffering from severe cardiovascular risk factors. Rejuvenating the reparative phenotype of autologous EPCs through a gene transfer approach has, therefore, been put forward as an alternative approach to enhance their therapeutic potential in cardiovascular patients. An increase in intracellular Ca2+ concentration constitutes a pivotal signal for the activation of the so-called endothelial colony forming cells (ECFCs), the only known truly endothelial EPC subset. Studies from our group showed that the Ca2+ toolkit differs between peripheral blood- and umbilical cord blood (UCB)-derived ECFCs. In the present article, we first discuss how VEGF uses repetitive Ca2+ spikes to regulate angiogenesis in ECFCs and outline how VEGF-induced intracellular Ca2+ oscillations differ between the two ECFC subtypes. We then hypothesize about the possibility to rejuvenate the biological activity of autologous ECFCs by transfecting the cell with the Ca2+ -permeable channel Transient Receptor Potential Canonical 3, which selectively drives the Ca2+ response to VEGF in UCB-derived ECFCs.

ID: 1020 Direct reprogramming of fibroblasts into endothelial progenitor cells by defined factors

Background: Endothelial progenitor cells (EPCs) are important progenitor cells in vasculogenesis as well as in tissue engineering. However, few EPCs can be isolated from bone marrow, peripheral blood and umbilical cord blood. Moreover, their in vitro proliferation potential is also limited. Therefore, this study aimed to produce EPCs from direct reprogramming of fibroblasts by transduction with certain specific factors.&#x0D; Methods: Human fibroblasts were collected from human skin by published protocols. The cells were transduced with 2 viral vectors containing 5 factors, including Oct3/4, Sox2, Klf4, c-Myc (plasmid 1), and VEGFR2 (plasmid 2). Transduced cells were treated with endothelial cell medium for 21 days. The cells were analyzed for expression of Oct3/4, Sox3, Klf4, c-Myc and VEGFR2 at day 5, and for EPC phenotype at day 21.&#x0D; Results: The results showed that after 5 days of transduction, fibroblasts acquired partial pluripotency. After 21 days of transduction and culture in endothelial cell medium, the cells exhibited endothelial markers (e.g. CD31 and VEGFR2) and formed blood vessel-like capillaries.&#x0D; Conclusion: Our findings suggest another strategy for direct reprogramming of fibroblasts into EPCs.

Erythropoietin-mediated activation of functional properties of peripheral blood mononuclear cells in patients with chronic heart failure

Stem cell therapy of diseases of the cardiovascular system, such as myocardial infarction is a prospective method for the stimulation of ischemic tissue repair. The main mechanisms of stem and progenitor cells action is a paracrine. The purpose of the study was to assess the effects of erythropoietin on the functional activity of mononuclear cells (MNCs) in patients with chronic heart failure before and after enrichment of peripheral blood with stem and progenitor cells mobilized by granulocyte colony-stimulating factor (G-CSF). 48 patients with coronary heart disease participated in the study. MNCs from the separated blood were isolated by density gradient on Ficoll/verografin. The phenotype of endothelial progenitor cells was investigated using monoclonal antibodies to CD34, CD133, VEGFR2, CD31. The Change of MNCs proliferative potential in response to erythropoietin was evaluated by MTT-test. The cytokine production in conditioned media was studied using ELISA. The effectiveness of mobilized MNC intramyocardial administration was assessed at 6 and 12 months by detection of a change in functional class according to NYHA heart failure, volume ejection fraction of the left ventricle of the heart and a change in myocardial perfusion. We showed that the enrichment of peripheral blood by mobilization of stem and progenitor cells in patients with chronic heart failure led to activation of proliferative potential of MNCs and increased erythropoietin production, a cytokine with pro-angiogenic activity. MNC enriched with stem and progenitor cells being culturing with erythropoietin increased the levels of TNF-α, IL-10, IL-18, IL-8, G-CSF and VEGF, as compared with the basal level of production. Circulating endothelial progenitor cells with the phenotype CD34- /VEGFR2+ have a correlation with the level of erythropoietin production. Secretory erythropoietin level directly correlated with myocardial perfusion, left ventricular ejection fraction and heart failure class at 6 and 12 months follow-up. The findings suggest that erythropoietin improves functional properties of the MNC of patients with heart failure after mobilization with G-CSF.

Evaluation of late outgrowth endothelial progenitor cell and umbilical vein endothelial cell responses to thromboresistant collagen-mimetic hydrogels.

Bioactive coatings which support the adhesion of late-outgrowth peripheral blood endothelial progenitor cells (EOCs) are actively being investigated as a means to promote rapid endothelialization of "off-the-shelf," small-caliber arterial graft prostheses following implantation. In the present work, we evaluated the behavior of EOCs on thromboresistant graft coatings based on the collagen-mimetic protein Scl2-2 and poly(ethylene glycol) (PEG) diacrylate. Specifically, the attachment, proliferation, migration, and phenotype of EOCs on PEG-Scl2-2 hydrogels were evaluated as a function of Scl2-2 concentration (4, 8, and 12 mg/mL) relative to human umbilical vein endothelial cells (HUVECs). Results demonstrate the ability of each PEG-Scl2-2 hydrogel formulation to support EOC and HUVEC adhesion, proliferation, and spreading. However, only the 8 and 12 mg/mL PEG-Scl2-2 hydrogels were able to support stable EOC and HUVEC confluence. These PEG-Scl2-2 formulations were, therefore, selected for evaluation of their impact on EOC and HUVEC phenotype relative to PEG-collagen hydrogels. Cumulatively, both gene and protein level data indicated that 8 mg/mL PEG-Scl2-2 hydrogels supported similar or improved levels of EOC maturation relative to PEG-collagen controls based on evaluation of CD34, VEGFR2, PECAM-1, and VE-Cadherin. The 8 mg/mL PEG-Scl2-2 hydrogels also appeared to support similar or improved levels of EOC homeostatic marker expression relative to PEG-collagen hydrogels based on von Willebrand factor, collagen IV, NOS3, thrombomodulin, and E-selectin assessment. Combined, the present results indicate that PEG-Scl2-2 hydrogels warrant further investigation as "off-the-shelf" graft coatings. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1712-1724, 2017.

Abstract 223: Brain-derived Neurotrophic Factor Promotes Regenerative Functions of Human Endothelial Progenitor Cells - Roles of miRNAs

Our previous study has demonstrated that brain-derived neurotrophic factor (BDNF) enhances anti-oxidant capacity of endothelial progenitor cells (EPCs). Since BDNF is produced in cerebrovascular endothelial cells, and functions as a vasoprotective molecule, we hypothesized that BDNF might enhance regenerative capacity of EPCs. Early EPCs (circulating angiogenic cells) were derived from healthy male subjects (48±1.5 years old, n=10). EPCs were treated with BDNF 100 ng/ml for 7 hours, and miRNA transcriptome was examined using Affymetrix miRNA array (version 3.0). BDNF significantly changed 31 miRNAs (n=4, P&lt;0.05, fold-change cut off=1.5). Notably, miR-181c level was increased by 1.9 fold (P&lt;0.05) after BDNF treatment. Since miR-181c is known to down-regulate TNF-α expression, BDNF may protect EPCs by promoting anti-inflammatory phenotype. We also found that BDNF decreased miR-433 level by 1.6 fold (P&lt;0.05). We further evaluated miR-433 function in human early EPCs. Knock-down of miR-433 in EPCs using siRNA significantly increased expression of guanylate binding protein 2 (GBP2) (n=6, P&lt;0.05), an established direct target for miR-433. In contrast, treatment with miR-433 mimic significantly reduced GBP2 protein expression in EPCs (n=4, P&lt;0.05). Since GBP2 has been shown to play an important role in the growth and colony formation of hematopoietic progenitors, BDNF/miR-433/GBP2 signaling may enhance angiogenic capacity of EPCs. In addition, neither miR-433-siRNA nor miR433 mimic significantly changed protein expressions of other targets such as growth factor receptor-binding protein2, cAMP response element-binding protein-1, cyclin-dependent kinase 6, and γ glutamyl cysteine synthetase (n=4-6, P&gt;0.05). In summary, changes in miRNA transcriptome induced by treatment with BDNF may promote anti-inflammatory and angiogenic phenotype of EPCs. These effects of BDNF may have important implications for therapeutic enhancement of regenerative capacity of EPCs.