Endothelial Progenitor Cells Colonies Research Articles

The Effect of SGLT2 Inhibitor Therapy on Endothelial Progenitor Cell Function in Patients with Heart Failure.

Sodium-glucose cotransporter-2 (SGLT-2) inhibitors have been shown to reduce the risk of cardiovascular mortality and hospitalizations in patients with heart failure (HF) with preserved or reduced ejection fraction (HFpEF or HFrEF). The mechanism for this benefit is not clear. Endothelial progenitor cells (EPCs) are bone-marrow derived cells able to differentiate into functional endothelial cells and participate in endothelial repair. The aim of the current study was to evaluate the effect of SGLT-2 inhibitors on the level and function of EPCs in patients with HF. We enrolled 20 patients with symptomatic HF, 12 with HFrEF and 8 with HFpEF (aged 73.3±10.2 years, 95% men). Blood samples were drawn at 2 time points: baseline and ≥3 months after initiation of SGLT-2 inhibitor therapy. Circulating EPC levels were evaluated by expression of VEGFR-2, CD34 and CD133 by flow-cytometry. EPCs colony forming units (CFUs) were quantified after 7 days in culture. The proportion of cells that co-expressed VEGFR-2 and CD34 or VEGFR-2 and CD133 was higher following 3 months of SGLT-2 inhibitors [0.26% (IQR 0.10-0.33) vs. 0.55% (IQR 0.28-0.91), P=0.002; 0.12% (IQR 0.07-0.15) vs. 0.24% (IQR 0.15-0.39), P=0.001, respectively]. EPCs-CFU were also increased following SGLT-2 inhibitor treatment [23 (IQR 3.7-37.8) vs. 79.4 (IQR 25.1-110.25) colonies/106 cells, P=0.0039]. In patients with symptomatic HF, both HFpEF and HFrEF, treatment with SGLT-2 inhibitors is associated with an increase in circulating EPCs level and function. This augmentation in EPCs may be a contributing mechanism to the clinical benefit of SGLT-2 inhibitors in HF patients.

(228) Cell-Based Therapy Can Restore Erectile Function in Men with Type 2 Diabetes and Metabolic Syndrome by Promoting Angiogenesis and Endothelial Repair

Abstract Introduction Although there has been a growing interest in restorative therapies for erectile dysfunction (ED) in recent years, there is still limited mechanistic evidence to support their effectiveness in the clinical setting. Objective The goal of this research was to investigate the mechanistic impact of cell-based therapy in men with ED secondary to Type 2 Diabetes Mellitus (DM2) and Metabolic Syndrome (MetS) through analysis of plasma biomarkers. Methods International Index of Erectile Function (IIEF) was completed by all men in the ACESO (NCT02886884) and CERES (NCT03059355) clinical trials. ACESO and CERES were both randomized, blinded, pilot phase clinical trials in which all participants received cellular therapy at varying doses and sources to treat endothelial dysfunction secondary to either DM2 or MetS, respectively. The intervention in both trials was peripheral intravenous infusion of allogeneic Mesenchymal Stem Cells (MSCs) of either 20 or 100 million cells derived from either bone marrow (BM) or umbilical cord tissue (UC). Follow-up was for 1 year. Endothelial function was measured via Brachial Artery Flow Mediated Vasodilation (FMD) and Endothelial Progenitor Cell-Colony Forming Units (EPC-CFU) assay. Plasma aliquots were isolated from blood samples and subjected to multiplex ELISA assay for analysis of angiogenic biomarkers. Results Data was pooled from all 20 men in the ACESO and CERES clinical trials. Overall, 7 received 20 million BM-MSCs, 9 received 100 million BM-MSCs, 2 received 20 million UC-MSCs, and 2 received 100 million UC-MSCs. There were no treatment emergent serious adverse effects (TE-SAEs) within 30 days following infusion in any of the participants. Among men with ED (IIEF-EF<25), 31.25% achieved MCID in erectile function. Participants with mild or moderate ED at baseline showed greater improvements in erectile function and endothelial function compared to those with severe ED. Participants who achieved MCID following cell therapy also experienced statistically significant improvements in FMD (P=0.05), whereas those who did not achieve MCID or had normal erectile function at baseline did not experience an improvement in FMD. Furthermore, men with Metabolic Syndrome who met MCID criteria were found to have increased Angiopoietin 1 and decreased Angiopoietin 2 following cell therapy. Conclusions The findings of this study demonstrate the potential of cell-based therapies in improving erectile function, angiogenic, and endothelial function. Future studies should aim to identify the causative mechanisms underlying these restorative effects in humans, paving the way for enhanced understanding and optimization of such therapies. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Longeveron LLC.

Effects of the factor Xa inhibitor rivaroxaban on the differentiation of endothelial progenitor cells

BackgroundWe evaluated the efficacy of the factor Xa inhibitor rivaroxaban on the differentiation ability of vascular endothelial progenitor cells (EPCs), which play roles in vascular injury repair and atherogenesis. Antithrombotic treatment in patients with atrial fibrillation undergoing percutaneous coronary intervention (PCI) is challenging, and current guidelines recommend oral anticoagulant monotherapy 1 year or more after PCI. However, biological evidence of the pharmacological effects of anticoagulants is insufficient.MethodsEPC colony-forming assays were performed using peripheral blood-derived CD34-positive cells from healthy volunteers. Adhesion and tube formation of cultured EPCs were assessed in human umbilical cord-derived CD34-positive cells. Endothelial cell surface markers were assessed using flow cytometry, and Akt and endothelial nitric oxide synthase (eNOS) phosphorylation were examined using western blot analysis of EPCs. Adhesion, tube formation and endothelial cell surface marker expression was observed in EPCs transfected with small interfering RNA (siRNA) against protease-activated receptor (PAR)-2. Finally, EPC behaviors were assessed in patients with atrial fibrillation undergoing PCI in whom warfarin was changed to rivaroxaban.ResultsRivaroxaban increased the number of large EPC colonies and increased the bioactivities of EPCs, including adhesion and tube formation. Rivaroxaban also increased vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, Tie-2, and E-selectin expression as well as Akt and eNOS phosphorylation. PAR-2 knockdown increased the bioactivities of EPCs and endothelial cell surface marker expression. Patients in whom the number of large colonies increased after switching to rivaroxaban showed better vascular repair.ConclusionsRivaroxaban increased the differentiation ability of EPCs, leading to potential advantages in the treatment of coronary artery disease.

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)

Abacavir antiretroviral therapy and indices of subclinical vascular disease in persons with HIV.

Indices of cardiovascular disease (CVD) risk, vascular endothelial dilation, arterial stiffness and endothelial repair were examined in persons with HIV (PWH) on an antiretroviral therapy (ART) that included abacavir (ABC+) in comparison with PWH on ART without abacavir (ABC-), and with HIV seronegative (HIV-) individuals. The 115 participants (63% men), aged 30-50 years, did not have CVD, metabolic, endocrine, or chronic renal conditions. PWH were on stable ART for six-months or more. Vascular assessments included flow-mediated dilation (FMD), aortic, radial and femoral arterial stiffness (cAIx, crPWV, cfPWV), and thigh and calf arterial compliance (Vmax50). Endothelial repair was indexed by endothelial progenitor cell colony forming units (EPC-CFU). Traditional CVD risk measures included blood pressure, central adiposity, lipids, insulin resistance (HOMA-IR), CRP and ASCVD score. Analyses controlled for demographics (age, sex, education), medications (antihypertensive, statin/fibrate, antipsychotic), and substance abuse (ASSIST). No group differences were observed in central adiposity, HOMA-IR, CRP, or ASCVD risk score. However, the ABC- group displayed greater dyslipidemia. The ABC+ group displayed no difference on FMD, cAIx, cfPWV or calf Vmax50 compared with other groups. When CD4 count and viral load were controlled, no additional differences between the ABC+ and ABC- groups emerged. Analyses of crPWV and thigh Vmax50 suggested supported by a trend toward lower EPC-CFU in the HIV+ groups than the HIV- group. Findings indicate that ABC treatment of 30-50 year-old PWH on stable ART is not likely to contribute in a robust way to higher CVD risk.

Abstract 10369: Artificial Intelligence-Assisted Prediction of Blood Cells Regenerative Capacity in Patients with Diabetes Mellitus

Introduction: Peripheral blood mononuclear cell (PBMC) regenerative capacity is impaired in pre/diabetes mellitus patients (DMPs). In vitro evaluation of PBMCs pre/post vasculogenic conditioning (VC) and integrative assessment of classical clinical risk factors using complex machine learning-based (ML) methods may help to distinguish patients' immune cell regenerative potential. Methods: Eighteen DMPs diagnosed with myocardial infarction and sixteen controls were enrolled. Isolated PBMC regenerative capability was evaluated pre/post-VC using endothelial progenitor cell colony formation assay/unit (EPC-CFA/U) and flow cytometry analysis. The following classification ML algorithms were used for data analysis: support vector machine (SVM), logistic regression (LR), random forest (RF), decision tree (DT), and adaptive boosting (AB). Results: An in vitro EPC-CFA assay revealed that DMPs showed decreased fresh PBMC-derived definitive EPCs (DEPCs) compared with controls. Decreased colony formation was observed in patients with a reduced number of circulating EPCs and they did not appropriately respond to VC according to ML findings. Furthermore, proinflammatory M1 macrophages significantly increased (P = 0.03) in the majority of DMPs (non-responders) compared to that in controls, whereas VC shifted the phenotype toward M2 along with CD4 + T and Treg cell expansion. Clinical, pre/post culture cell phenotype, and EPC-CFU data integration and comprehensive ML analyses distinguished “non-responder”-associated distinct biomarkers, clinical parameters, such as elevated proinflammatory cells and systemic inflammation, and elderly patients from those of responders. Notably, SVM prediction accuracy was 100%; both LR and RF were 86%; and those of AB and DT were 71% and 43%, respectively. Conclusions: Applying comprehensive ML methods, as well as integrative analysis of the findings of in vitro assays, such as EPC-CFA; such as EPCs, M1/M2 macrophages, and T cells pre/post-VC; and clinical-laboratory parameters, enabling us to precisely evaluate regeneration capacity of impaired PBMNC functions. Moreover, this cell culture methodology can be used before cell therapy to define optimal dose and cell ratio for transplantation.

Abstract 14353: Feminizing Hormone Therapy and Subclinical Indicators of CVD Risk in Transgender Women With HIV

Introduction: Transgender women (TW) are at higher risk of HIV infection compared to cisgender groups and HIV is associated with accelerated cardiovascular disease (CVD) complications. A high proportion of TW use feminizing hormone therapy (FHT), particularly estrogen. However, there is a scarcity of data about the cardiovascular impact of FHT in TW with HIV (TWH). Subclinical indicators of CVD risk, arterial stiffness and endothelial progenitor cells colony forming units (EPC-CFUs), were examined in TWH with and without FHT. Hypothesis: Estrogen use in FHT reduces arterial stiffness and induces EPC proliferation. Methods: This pilot included adults 20-60 years old, identifying as TW, with a documented HIV-1 diagnosis, stable ART ≥ 6 months, and without history of CVD. Sociodemographic questionnaire was completed prior to an in-person blood collection and applanation tonometry assessment. Groups were stratified by estrogen levels (normal: 1-82pg/ml vs high: >82pg/ml) and relationships with EPC-CFUs and arterial stiffness were examined using t-test and Pearson’s correlation. Results: Median age was 42 yrs. (IQR 34-52) with mean glucose 105.70 mg/dl (± 58.07), triglycerides 138.52 mg/dl (± 69.61), LDL 82.38 mg/dl (± 35.01), and CRP 3.45 mg/L (±3.57). TW with high (n=5) and normal (n=12) estrogen levels had similar values in EPC-CFUs counts (p=0.99), central augmentation index (cAIx; p=0.27), carotid to radial pulse wave velocity (CR-PWV; p=0.20), and carotid to femoral PWV (CF-PWV; p=0.87). A positive trend appeared in log-transformed estradiol across cAIx (r=0.32, p=0.21); negative trends in CR-PWV (r=-0.11, p=0.70) and CF-PWV (r=-0.19, p=0.48), and no relationship with EPC-CFUs (r=0.05, p=0.86). There were non-significant negative weak trends between EPC-CFUs and cAIx (r=-0.32, p=0.21), CR-PWV (r=-0.2, p=0.48), and CF-PWV (r =-0.29, p=0.27). Conclusions: An inverse correlation between EPC-CFUs and arterial stiffness measurements was observed. Considering these test modalities have a strong correlative potential with CVD risk, FHT does not appear to confer higher CVD risk through these mechanisms. Further research to understand the mechanisms underlying HIV related CVD pathophysiology is critically needed especially in TWH.

Isolating and characterizing lymphatic endothelial progenitor cells for potential therapeutic lymphangiogenic applications

Lymphatic dysfunction is associated with the progression of several vascular disorders, though currently, there are limited strategies to promote new lymphatic vasculature (i.e., lymphangiogenesis) to restore lost lymphatic function. One promising approach to stimulate lymphangiogenesis involves delivering endothelial progenitor cells (EPCs), which are naturally involved in de novo blood vessel formation and have recently been identified to include a lymphatic subpopulation. However, the contribution of lymphatic EPCs in lymphangiogenesis is not clear and challenges with maintaining the activity of transplanted EPCs remain. Thus, the objective of this study was to isolate lymphatic EPCs from human umbilical cord blood and characterize their role in the initial stages of blood or lymphatic vasculature formation. Furthermore, this study also tested the applicability of alginate hydrogels to deliver lymphatic EPCs for a possible therapeutic application. We postulated and confirmed that blood and lymphatic EPC colonies could be isolated from human umbilical cord blood. Additionally, EPC populations responded to either angiogenic or lymphangiogenic growth factors and could stimulate their respective mature endothelial cells in vasculature models in vitro. Finally, lymphatic EPCs maintained their ability to promote lymphatic sprouts after prolonged interactions with the alginate hydrogel microenvironment. These results suggest EPCs have both a blood and a lymphatic population that have specific roles in promoting revascularization and highlight the potential of alginate hydrogels for the delivery of lymphatic EPCs. Statement of significanceDespite the potential therapeutic benefit of promoting lymphatic vasculature, lymphangiogenesis remains understudied. One appealing strategy for promoting lymphangiogenesis involves delivering lymphatic endothelial progenitor cells (EPCs), which are a subpopulation of EPCs involved in de novo vessel formation. Here, we investigate the role of isolated blood and lymphatic EPC subpopulations in promoting the early stages of vascularization and the utility of alginate hydrogels to deliver lymphatic EPCs. We determined that EPCs had two populations that expressed either blood or lymphatic markers, could stimulate their respective mature vasculature in tissue constructs and that alginate hydrogels maintained the therapeutic potential of lymphatic EPCs. We anticipate this work could support promising biomaterial applications of EPCs to promote revascularization, which could have many therapeutic applications.

Quality and Quantity-Cultured Human Mononuclear Cells Improve Human Fat Graft Vascularization and Survival in an In Vivo Murine Experimental Model.

Fat graft ischemia impedes us from having satisfying long-term results. The quality and quantity culture is a 1-week cell culture that increases the vasculogenic potential of peripheral blood mononuclear cells (PBMNC). This in vivo murine model investigates whether enrichment with quality and quantity-cultured human mononuclear cells (MNC-QQ) improves the vascularization in the human fat graft and whether this decreases the tissue loss. Human adipose tissue, PBMNC, MNC-QQ, and stromal vascular fraction were prepared. First, PBMNC, MNC-QQ, and stromal vascular fraction were compared in vitro for vasculogenic potential by endothelial progenitor cell colony-forming and culture assays. Second, 0.25-g fat grafts were created with 1 × 106 PBMNC (n = 16), 1 × 106 MNC-QQ (n = 16), 1 × 106 stromal vascular fraction (n = 16), or phosphate-buffered saline as control (n = 16) before grafting in BALB/c nude mice. Grafts were analyzed for weight persistence, vessel formation by CD31 immunohistochemistry, and angiogenic markers by quantitative polymerase chain reaction. MNC-QQ develop more definitive endothelial progenitor cell colonies and more functional endothelial progenitor cells compared to PBMNC and stromal vascular fraction. Weight persistence after 7 weeks was significantly higher in grafts with MNC-QQ (89.8 ± 3.5 percent) or stromal vascular fraction (90.1 ± 4.2 percent) compared with control (70.4 ± 6.3 percent; p < 0.05). MNC-QQ-enriched grafts had the highest vessel density (96.6 ± 6.5 vessels/mm2; control, 70.4 ± 5.6 vessels/mm2; p < 0.05). MNC-QQ exerted a direct vasculogenic effect through vascular integration and a potential paracrine vascular endothelial growth factor-mediated effect. Quality and quantity-cultured human mononuclear cells containing endothelial progenitor cells stimulate fat graft vascularization and enhance graft survival in a rodent recipient.

Garlic Extract (Allicin) Improves the Proliferation of Endothelial Progenitor Cell (EPC) from Patients with Stable Coronary Artery Disease

BACKGROUND: The reduced number and function of endothelial progenitor cell (EPC) in stable coronary artery disease (SCAD) patients aggravate endothelial dysfunction and inhibit neovascularization, thus lead to atherosclerosis. Garlic is currently believed to increase the number and function of EPC.&#x0D; AIM: Therefore, this in vitro study was conducted to analyze the effect of garlic extract (allicin) on the proliferation of EPC in patients with SCAD.&#x0D; METHODS: Mononuclear cells were isolated from peripheral blood of eight SCAD patients and cultured on colony-forming unit (CFU)-Hill medium for 3 days. Samples were divided into two groups: Group treated with allicin and control group. The treatment group was then divided into three subgroups which received 10, 50, and 100 mg/ml of doses and incubated for 48 h. EPC proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. Immunohistochemical method of CD34+ was performed for EPC identification. Data were analyzed using independent t-test and ANOVA.&#x0D; RESULTS: MTT assay showed a significant increase in EPC proliferation in the allicin group compared to the control group (0.2811 ± 0.008 vs. 0.194 ± 0.151, p &lt; 0.05) and significant improvements were observed in each dose increment. CFU-Hill quantification shows the addition of EPC colony in high-dose allicin. Immunohistochemical method shows positive CD34+ expression.&#x0D; CONCLUSION: Allicin increases EPC proliferation dose-dependently from peripheral blood of SCAD patients.

Effects of Garlic Extract (allicin) on Proliferation of Endothelial Progenitor Cells (EPC) in Patients with Stable Coronary Artery Disease

The reduced number and function of Endothelial Progenitor Cells (EPC) in stable coronary artery disease (SCAD) patients aggravate endothelial dysfunction and inhibit neovascularization, thus leading to atherosclerosis. Garlic is currently believed to increase the number and function of EPCs. Therefore, this in vitro study was conducted to analyse the effect of garlic extract (Allicin) on the proliferation of EPCs in patients with SCAD. Mononuclear cells were isolated from peripheral blood of eight SCAD patients and cultured on CFU-Hill media for three days. Samples were divided into 2 groups: a group treated with Allicin and a control group. The treatment group was then divided into 3 subgroups which received 10, 50, and 100 mg/ml of doses and incubated for 48 hours. EPC proliferation was assessed using MTT Cell Proliferation Assay. Immunohistochemical method of CD34+ were performed for EPC identification. Data was analysed using an independent T test and ANOVA. MTT Assay showed significant increase in EPC proliferation in Allicin group compared to control group (0.2811±0.008 vs 0.194±0.151, p<0.05) and significant improvements were observed in each dose increment. CFU-Hill quantification shows the addition of EPC colony in high-dose Allicin. Immunohistochemical method shows positive CD34+ expression. Allicin increases EPC proliferation dose-dependently from peripheral blood of SCAD patients.

310 Does Improvement in Endothelial Function affect Erectile Function following Stem Cell Therapy in Men with Cardiomyopathy?

Endothelial dysfunction is an important component in the pathogenesis of cardiovascular disease and erectile dysfunction (ED). Interest in cell-based therapy (CBT) for cardiovascular disease and ED is growing. Early trials have shown CBT to improve cardiac function in men with ischemic cardiomyopathy, with concurrent improvement in endothelial function. Whether or not the improvement in endothelial function from CBT may benefit ED has yet to be explored. To determine if a change in endothelial function affects erectile function in men who received transendocardial injection of stem cells. We performed a post hoc sub-analysis of the TRIDENT trial to examine the relationship between endothelial function and erectile function. The TRIDENT study recruited 30 participants with ischemic cardiomyopathy and reduced ejection fraction (<50%) to receive allogenic bone-marrow derived mesenchymal stem cells (MSC) via transendocardial injection (TESI). Participants were randomized to receive MSCs in doses of 20 or 100 million cells and were followed for 1 year. International Index of Erectile Function (IIEF) questionnaires were administered at baseline and at 3, 6 and 12 months. Endothelial function was assessed at baseline and 3 months post-therapy and consisted of two measures: brachial artery percent Flow Mediated Vasodilation (FMD) and quantification of Endothelial Progenitor Cell-Colony Forming Units (EPC-CFUs). Patients with FMD less than 7% were considered to have endothelial dysfunction (normal = >10%), and higher counts of circulating EPCs indicate a greater capacity for endothelial self-regeneration.

B lymphocyte stimulator modulates number and function of endothelial progenitor cells in systemic lupus erythematosus

BackgroundCirculating endothelial progenitor cells (EPCs) are biologic markers of endothelial function. In patients with systemic lupus erythematosus (SLE), the numerical reduction and functional impairment of EPCs contribute to the endothelial dysfunction.Through ex vivo and in vitro studies, we aimed at evaluating the effects of B lymphocyte stimulator (BLyS) on EPC colonies and endothelial cells and also investigating BLyS receptor expression on these cells.MethodsEPCs were isolated from peripheral blood mononuclear cells (PBMC). In order to evaluate their ability to form colonies, EPCs were cultured on fibronectin-coated dishes and incubated with BlyS alone or BlyS and belimumab. Apoptosis of EPCs and endothelial cell line EA.hy926 was evaluated after 6, 12, and 24 h of incubation with BLyS and after 6 h with BLyS and belimumab. The expression of B cell activating factor-receptor (BAFF-R), B cell maturation antigen (BCMA), and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI) on EPCs and EA.hy926 was analyzed by cytofluorimetry.ResultsThe number of EPC colonies was lower in patients than in controls. Moreover, the colonies from SLE patients were poorly organized compared to controls; the addition of belimumab restored the colony structure. Incubation with BLyS induced apoptosis of EPCs and EA.hy926 that was inhibited by the co-incubation with belimumab. BAFF-R and BCMA were expressed on both EPCs and EA.hy926, while TACI was expressed only on EPCs.ConclusionsEPCs and endothelial cells preferentially express BAFF-R which could be involved in the pro-apoptotic effect of BlyS. Belimumab administration seems to restore the quantitative and qualitative changes of EPC colonies both ex vivo and in vitro.

Insufficient production of IL-10 from M2 macrophages impairs in vitro endothelial progenitor cell differentiation in patients with Moyamoya disease

Moyamoya disease (MMD) is well known to be caused by insufficient cerebral vascular formation. However, the essential pathogenesis has not yet been identified. Using our recently developed technique of generating vasculogenic and anti-inflammatory cultures, we investigated endothelial progenitor cell (EPC) expansion and differentiation under the cytokine milieu generated by the peripheral blood mononuclear cells (PBMNCs) of the operated and non-operated MMD patients. EPC colony forming assay of the cultured PBMNCs disclosed the decline of the definitive EPC colony numbers in the both MMD patients. The level of interleukin-10 (IL-10) was lower in secretory cytokines from the cultured PBMNCs of MMD patients than that in that of controls using a cytometric bead array. The addition of human recombinant IL-10 to PBMNCs cultured from MMD patients restored the EPC colony forming potential of MMD PBMNCs. Following phorbol myristate acetate stimulation of the cultured PBMNCs, flow cytometry revealed a decrease in intracellular IL-10 storage in the main cell populations of the PBMNCs cultured from MMD patients relative to those cultured from controls. The present data provide the expected mechanism of vascular malformation in MMD pathogenesis originated from the insufficient production of IL-10 secreting cells from PBMNCs fostering EPC expansion and differentiation.

Physical activity intervention improved the number and functionality of endothelial progenitor cells in low birth weight children

Background and aimsThe purpose of this study was to investigate whether an intervention with physical activity (PA) would promote positive effects on the angiogenic factors, mobilization, and functionality of circulating endothelial progenitor cells (EPCs) in children with low birth weight (LBW). Methods and resultsThirty-five children participated in a 10-week PA program (intensity: 75–85% of heart rate reserve, frequency: four times/week, and duration: 45 min). Before and after the PA program, we evaluated anthropometric parameters, blood pressure levels, biochemical profile, number of EPCs, number of EPC colony forming units, and plasma levels of vascular endothelial growth factor-A (VEGF-A), nitric oxide (NO), and matrix metalloproteinases (MMPs) 2 and 9. We found a significant main effect of the PA program on waist circumference (ηp2 = 0.489), cardiorespiratory fitness (ηp2 = 0.463), and MMP-9 (ηp2 = 0.582). Birth weight or the PA program produced significant independent effects on systolic blood pressure (birth weight: ηp2 = 0.431; PA program: ηp2 = 0.615) and EPC colony forming units (birth weight: ηp2 = 0.541; PA program: ηp2 = 0.698) with no significant interactions. The combination of birth weight and the PA program produced a significant interaction effect on the number of circulating EPCs (ηp2 = 0.123), NO (ηp2 = 0.258), and VEGF-A (ηp2 = 0.175). The variation in the number of EPCs from baseline to 10 weeks of the PA program correlated positively with the change in NO (P = 0.002) and VEGF-A (P = 0.004). ConclusionsA 10-week PA program attenuates the adverse effect of LBW on the number and functionality of EPCs; this effect occurs through an improvement in circulating levels of NO and VEGF-A. Clinical trialshttps://www.clinicaltrials.gov. Unique Identifier: NCT02982967. Date: December/2016.

Effects of metoprolol, methyldopa, and nifedipine on endothelial progenitor cells in patients with gestational hypertension and preeclampsia

Endothelial progenitor cells (EPCs) are critical for vascular regeneration and function, but are reduced in hypertensive disorders of pregnancy. We aimed to determine the possible effects of antihypertensive drugs, such as metoprolol, methyldopa, and nifedipine, on EPC number and functions in patients with gestational hypertension and preeclampsia. We collected blood samples from 30 normal pregnant women, 67 patients with gestational hypertension and 48 patients with preeclampsia. The patients received no drug or an antihypertensive drug, such as metoprolol, methyldopa, or nifedipine, between 20 and 24weeks of gestation. The number of EPCs and circulating endothelial cells (CECs) in the blood was measured by flow cytometry. Moreover, colony formation and migration assays were performed on the isolated EPCs. Both the systolic and diastolic blood pressure (BP) increased, while the percentage of flow-mediated vasodilatation (FMD) decreased in patients with gestational hypertension and preeclampsia, compared to the healthy controls at 20weeks of gestation. CEC number increased in the patients, whereas EPC counts decreased. Furthermore, EPC colony formation and migration abilities were also impaired in the patients. However, administration of metoprolol, methyldopa, or nifedipine effectively restored the systolic and diastolic BP, FMD%, EPCs, and CEC numbers, as well as EPC migration capacity. Endothelial progenitor cells colony formation ability selectively improved with methyldopa and nifedipine. In patients receiving no drugs, most of these indexes worsened within 4weeks (study duration). This study revealed a new pharmacological action of these antihypertensive drugs against gestational hypertension and preeclampsia, thus supporting their clinical use.

Regeneration-associated cell transplantation contributes to tissue recovery in mice with acute ischemic stroke.

Various cell-based therapeutic strategies have been investigated for vascular and tissue regeneration after ischemic stroke. We have developed a novel cell population, called regeneration-associated cells (RACs), by quality- and quantity-controlled culture of unfractionated mononuclear cells. RACs were trans-arterially injected into 10-week-old syngeneic male mice at 1, 3, 5 or 7 days after permanent middle cerebral artery occlusion (MCAO) to determine the optimal timing for administration in terms of outcome at day 21. Next, we examined the effects of RACs injection at day 1 after MCAO on neurological deficits, infarct volume, and mediators of vascular regeneration and anti-inflammation at days 7 and 21. Infarct volume at day 21 was significantly reduced by transplantation of RACs at day 1 or 3. RACs injected at day 1 reduced the infarct volume at day 7 and 21. Angiogenesis and anti-inflammatory mediators, VEGF and IL-10, were increased at day 7, and VEGF was still upregulated at day 21. We also observed significantly enhanced ink perfusion in vivo, tube formation in vitro, and definitive endothelial progenitor cell colonies in colony assay. These results suggest that RAC transplantation in MCAO models promoted significant recovery of neural tissues through intensified anti-inflammatory and angiogenic effects.

Humoral and Cellular Patterns of Early Endothelial Progenitor Cells in Relation to the Cardiovascular Risk in Axial Spondylarthritis.

BackgroundSpondylarthritis (SpA) significantly affects sacroiliac, intervertebral and peripheral joints. Patients with SpA suffer from increased cardiovascular risk (CVR). The endothelial progenitor cell (EPC) system critically perpetuates vascular repair. The aim of the study was to evaluate circulating EPCs in axial (ax)SpA with special attention on parameters of disease activity and CVR.MethodsDisease activity and functional impairment were quantified in 50 axSpA patients by using standardized parameters (Bath ankylosing spondylitis disease activity index (BASDAI), C-reactive protein (CRP), finger-floor distance (FFD) and Ott’ sign). Circulating EPCs and EPC regeneration were analyzed (fluorescence-activated cell sorting (FACS) and colony-forming unit (CFU) assay). Serum vasomodulatory mediators were quantified by enzyme-linked immunosorbent assay (ELISA).ResultsEPC colony numbers were lower in axSpA as compared to controls. Females displayed more colonies than males. In addition, fewer colonies were observed in smokers, in patients with a BASDAI of below 4 and in hypertension. Circulating CD133+/KDR+ cells did not differ between the groups. Follow-up analysis (33 months later) did not show any differences in gender, colony formation, CD133+/KDR+ cells or serum levels of vasomodulatory mediators if related to the categories of BASDAI, Ott’ sign or FFD.ConclusionsEPC colony formation is significantly affected in axSpA with particularly low levels in males. EPC-related parameters do not allow predicting disease activity-related or functional parameters nor are they useful for CVR assessment in SpA.

Osteoprotegerin Induces CD34+ Differentiation in Endothelial Progenitor Cells.

Endothelial progenitor cells (EPCs) are the main hypothetical cells that could give rise to vessels and in particular one subtype isolated from peripheral or cord bloods: endothelial colony forming cells (ECFCs). These ECFCs are clonogenic precursors committed to endothelial lineage and have robust vasculogenic properties. However, their low number and poor expansion properties when isolated from human adult bloods, currently limit their use as an autologous cell therapy product. We previously reported that osteoprotegerin (OPG), a well-characterized regulator of bone metabolism, contributes to ischemic tissue revascularization, tumor growth in vivo, and potentiates ECFCs proangiogenic properties through the secretion of SDF-1. The current study investigated the role of OPG in ECFCs differentiation and expansion from cord blood CD34+ cells. OPG increased the number of ECFCs after endothelial differentiation of CD34+ cells, enhancing the time of EPCs colonies initial appearance and the growth kinetic of endothelial cell progeny. OPG-exposed ECFCs expressed higher levels of CD34+ compared to control ECFCs. In conclusion, our findings provide novel insights into OPG in regulation of CD34+ progenitor cells. These results give new opportunities for ex vivo expansion of human ECFCs using OPG as a cell culture component for future ECFC product manufacture according to GMP.

Dysfunctional high density lipoprotein failed to rescue the function of oxidized low density lipoprotein-treated endothelial progenitor cells: a novel index for the prediction of HDL functionality

Lipid metabolic disorders play critical roles in atherogenesis. Traditionally, it has been suggested that reduced high density lipoprotein (HDL) levels might be an important morbidity indicator for cardiovascular diseases. Therefore, it has been argued that therapeutically raising HDL levels may reduce atherogenesis in patients with dyslipidemia. However, recent clinical trials to elevate serum HDL levels by pharmacologic approaches failed to demonstrate clinical efficacy. Thus, to investigate the functionality of HDL and to explore the possible clinical relevance as well as to define an effective indicator that can represent HDL function may provide another key and reference to disclose the clinical treatment of dyslipidemia. We analyzed the association between the data of dichlorofluorescein assay (assay the functionality of HDL), the effect of HDL on oxidized low density lipoprotein (oxLDL)-stimulated endothelial progenitor cells (EPCs) in vitro, levels of circulating EPCs, and ex vitro EPC colony forming units of each case, we defined the indicator (relative HDL index (RHDL index) = dichlorofluorescein assay result of each subject/dichlorofluorescein assay reading of our young healthy controls) that may represent functionality of HDL. HDL from healthy adults protected oxLDL-treated EPCs by modulating p38 mitogen-activated protein kinase and Rho activation and by promoting nitric oxide production. HDL from subject with RHDL index ≧2 also failed to restore the functionality of oxLDL-treated EPCs via cell-signaling pathways in vitro. The RHDL index significantly correlated with patients' circulating EPC number or EPC colony forming units ex vivo. In conclusions, we explored the RHDL index as a score to predict a patient's EPC functions in vivo and ex vitro.