Endothelial Progenitor Cells Colony-forming Units 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 this 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 vascular endothelial growth factor receptor-2 (VEGFR-2), CD34, and CD133 by flow cytometry. EPC colony forming units (CFUs) were quantified after 7 days in culture. The proportion of cells that coexpressed VEGFR-2 and CD34 or VEGFR-2 and CD133 was higher following 3 months of SGLT-2 inhibitors [0.26% (interquartile range, IQR 0.10-0.33) versus 0.55% (IQR 0.28-0.91), P = 0.002; 0.12% (IQR 0.07-0.15) versus 0.24% (IQR 0.15-0.39), P = 0.001, respectively]. EPC CFUs were also increased following SGLT-2 inhibitor treatment [23 (IQR 3.7-37.8) versus 79.4 (IQR 25.1-110.25) colonies/10 6 cells, P = 0.0039]. In patients with symptomatic HF, both HFpEF and HFrEF, treatment with SGLT-2 inhibitors is associated with an increase in the level and function of circulating EPCs. This augmentation in EPCs may be a contributing mechanism to the clinical benefit of SGLT-2 inhibitors in patients with HF.

(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.

Ramipril Improves Endothelial Function and Increases the Number of Endothelial Progenitor Cells in Patients With Systemic Lupus Erythematosus.

Endothelial dysfunction and reduced number of endothelial progenitor cells (EPCs) in peripheral blood are contributing factors to cardiovascular disease in systemic lupus erythematosus (SLE) patients. Endothelial progenitor cell proliferation is regulated by vascular endothelial growth factor (VEGF). Angiotensin-converting enzyme inhibitors reduce cardiovascular mortality in patients with coronary heart disease. This was a randomized trial including 37 female SLE patients without cardiovascular risk factors allocated into 2 groups: 19 patients received ramipril 10 mg/d for 12 weeks (IG) and 18 patients maintained without ramipril (CG). Endothelial function was assessed by brachial artery ultrasound measuring flow-mediated dilation, and EPCs were quantified by flow cytometry and cell culture, at baseline and after 12 weeks. Serum VEGF levels were measured by enzyme-linked immunosorbent assay. Statistical analysis was intention to treat. p < 0.05 was considered significant. After 12 weeks, higher flow-mediated dilation (6.17% vs. 11.14%, p < 0.001) was observed in IG, without change in CG (5.37% vs. 5.02%, p = 0.630). Higher number of EPC colony-forming units was also observed in IG (21.3 ± 10.4 vs. 31.6 ± 8.5, p < 0.001), without difference in CG ( p = 0.714). No difference was found in EPCs evaluated by flow cytometry. Vascular endothelial growth factor level increased after 12 weeks in IG ( p = 0.048), with no difference in CG ( p = 0.661). Ramipril improved endothelial function and increased the numbers of EPCs evaluated by cell culture and VEGF levels in SLE patients without cardiovascular risk factors. These data suggest that angiotensin-converting enzyme inhibitor bring an extra benefit beyond the hypotensive action and should be considered as a preferred antihypertensive drug in SLE patients.

Could COVID-19 Induced Endothelial Dysfunction Explain New Onset Peyronie's Disease?

IntroductionCoronavirus Disease 2019 (COVID-19) is an emerging infectious disease caused by a novel coronavirus (SARS-CoV-2) which has been found to cause systemic inflammation in some patients. Many possible long-term sequelae of COVID-19 have yet to be identified. Since endothelial cells have both angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2) receptors, and SARS-CoV-2 requires both receptors for cell entry, we hypothesized that widespread endothelial damage caused by COVID-19 infection leads to Peyronie's disease (PD).ObjectiveNew-onset PD after resolved COVID-19 infection was evaluated.MethodsThis study was performed after Institutional Review Board approval. After comprehensive history and physical exam, penile duplex was used to evaluate blood flow and characterize any plaques. International Index of Erectile Function (IIEF) was used to evaluate erectile function. Brachial artery flow-mediated dilation (FMD) and endothelial progenitor cell – colony forming units (EPC-CFU) assays were utilized as measures of endothelial function. Available data from two separate control groups were used for comparisons: “No PD” contains subjects with a history of cardiovascular disease and normal erectile function according to IIEF-EF score (n=27). This group was selected to serve as a negative control with endothelial dysfunction. A second control group “Healthy Controls” was selected as a positive control containing male participants who were free from any significant cardiovascular medical history (n=9).ResultsNew-onset PD after resolution of COVID-19 infection was identified in one patient. The penile duplex with erectogenic agents demonstrated normal vascular flow with presence of calcified plaques. The PD group median EPC-CFU levels were 0 as compared to 1.60 in the no PD (negative control) group and 20.00 in healthy (positive) controls. Similarly, median FMD% was impaired in the PD group (2.35%) and no PD group (3.88%) as compared to healthy controls (7.75%).ConclusionsThese findings reveal a novel mechanism for PD after resolution of COVID-19 infection. These findings have significant clinical and biological implications for males with PD and should be considered as a possible sequela of COVID-19 infection.DisclosureAny of the authors act as a consultant, employee or shareholder of an industry for: Acerus Pharmaceuticals, Boston Scientific, Coloplast, Endo Pharmaceuticals, Empower Pharmacy, Nestle Health, Olympus

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 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: &gt;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.

Abstract 9813: A Randomized, Double Blind, Clinical Trial to Evaluate Safety and Efficacy of Allogeneic Mesenchymal Human Stem Cells Infusion Therapy for Endothelial Dysfunction in Diabetic Subjects (ACESO)

Introduction: Endothelial dysfunction underlies the pathophysiology of cardiovascular disease (CVD) in patients with diabetes mellitus (DM), but no known effective therapies exist. The randomized, double-blind ACESO clinical trial evaluated the safety and efficacy of allogeneic human mesenchymal stem cells (allo-MSCs) infusion to target endothelial dysfunction in Type 2 DM subjects. Methods: An open label pilot phase (n=6) was followed by a blinded, randomized phase (n=10) study. DM subjects with endothelial dysfunction received a single peripheral intravenous infusion of either 20 or 100 million allo-MSCs (obtained from 3 healthy bone marrow donors) and followed for 1 yr. The primary endpoint was incidence of treatment-emergent serious adverse events (TE-SAE) within 1-month post-infusion. Secondary endpoints assessed endothelial function via endothelial progenitor cell-colony forming units (EPC-CFUs) and flow-mediated dilation (FMD%). Results: Participants had well-controlled DM, blood pressure, and kidney function (GFR ≥50 ml/min), and most were taking statins. The 6 pilot-phase participants were men (1 Asian, 5 non-Hispanic white) with mean (±SD) age of 69.3 (9.6) years. The 10 randomized participants were 5 men and 5 women (5 Hispanic, 3 non-Hispanic Black), with mean age of 60.9 (10.6) years. No TE-SAEs were reported. None of the patients in the pilot phase developed Panel Reactive Antibodies (PRAs). One randomized subject had a moderate increase in PRAs after infusion. Both treatment groups (20 and 100M allo-MSCs) in the pilot and randomized phases showed increased EPC-CFUs over time, particularly at 14 and 28 days post-infusion, that was sustained at 6-12 months. FMD data followed a trend rising above baseline and peaking at days 7 to 14, and then subsided to pre-infusion levels by 1 yr. Conclusion: Peripheral infusion of allo-MSCs for patients with DM and endothelial dysfunction is safe. Allo-MSCs improve EPCs and FMD within 14 to 28 days post-infusion, and the effect is sustained for 6 to 12 months. Larger clinical trials are warranted to assess the efficacy of allo-MSC therapy for cardiovascular outcomes in diabetic patients. Clinical Trial Registration: URL: https://www.clinicaltrials.gov.; Unique ID: NCT02886884

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.

Mesenchymal Stem Cell Secretion of SDF-1α Modulates Endothelial Function in Dilated Cardiomyopathy.

BackgroundEndothelial dysfunction contributes to the pathophysiology of dilated cardiomyopathy (DCM). Allogeneic but not autologous mesenchymal stem cells (MSCs) improve endothelial function in DCM patients. We hypothesized that these effects are modulated by release of stromal derived factor-1α (SDF-1α).MethodsPlasma TNFα and endothelial progenitor cell-colony forming units (EPC-CFUs) were assessed at baseline and 3-months post-injection in a subset of POSEIDON-DCM patients that received autologous (n = 11) or allogeneic (n = 10) MSCs. SDF-1α secretion by MSCs, endothelial cell (EC) TNFα mRNA expression, and levels of reactive oxygen species (ROS) in response to SDF-1α were measured in vitro.ResultsAs previously shown, DCM patients (n = 21) had reduced EPC-CFUs at baseline (3 ± 3), which were restored to normal by allogeneic MSCs 3-months post-treatment (Δ10 ± 4). DCM patients had elevated baseline plasma TNFα (n = 15, 22 ± 9.4 pg/mL). Allogeneic MSCs (n = 8) decreased, and autologous MSCs (n = 7) increased, plasma TNFα (−7.1 ± 3.1 vs. 22.2 ± 17.1 pg/mL, respectively; P = 0.0005). In culture, autologous MSCs (n = 11) secreted higher levels of SDF-1α than allogeneic MSCs (n = 6) [76.0 (63.7, 100.9) vs. 22.8 (7.2, 43.5) pg/mL, P = 0.0002]. SDF-1α and plasma TNFα negatively correlated with EPC-CFUs in both treatment groups (R = −0.7, P = 0.0004). ECs treated with 20 ng SDF-1α expressed lower levels of TNFα mRNA than cells treated with 100 ng (0.7 ± 0.2 vs. 2.1 ± 0.3, P = 0.0008). SDF-1α at low but not high concentration inhibited the generation of ROS.ConclusionMSC secretion of SDF-1α inversely correlates with EPC-CFU production in DCM patients and therefore may be a modulator of MSC therapeutic effect in this clinical setting.Clinical Trial Registrationhttps://clinicaltrials.gov/ct2/show/NCT01392625, identifier NCT01392625.

Beneficial Impact of Moderate to Vigorous Physical Activity Program on Circulating Number and Functional Capacity of Endothelial Progenitor Cells in Children: The Crucial Role of Nitric Oxide and VEGF-A.

Endothelial progenitor cells (EPCs) appear to interact with physical training. This study aimed to provide a comprehensive assessment of the relationship of moderate to vigorous physical activity (MVPA) with both angiogenic factors and EPC function in healthy children. Forty children (22 boys and 18 girls) aged 7 to 11 years participated in a 10-week MVPA program (duration: 45min; intensity: 75%-85% of heart rate reserve; frequency: 4sessions/wk). The anthropometric data, biochemical profile, EPCs number, EPCs colony-forming units, and vascular endothelial growth factor-A (VEGF-A) and nitric oxide (NO) plasma levels were evaluated before and after the MVPA program. After a 10-week MVPA program, a significant increase was detected in circulating/functional capacity of EPCs, NO, and VEGF-A levels, associated with improvement of waist circumference and estimated maximum rate of oxygen consumption (VO2max). A strong positive correlation was found between delta of EPCs number and variation of both NO level (r = .677, P < .001) and VEGF-A level (r = .588, P < .001). Furthermore, a significant correlation between NO level variation and delta of VEGF-A level was observed (r = .708, P < .001). Our findings suggest that lifestyle intervention implemented by MVPA program can contribute meaningfully to improve circulating/functional capacity of EPCs in healthy children, possibly due to the increase of plasma NO and VEGF-A levels.

Berberine reduces endothelial injury and arterial stiffness in spontaneously hypertensive rats

ABSTRACTBackground: Changes in circulating endothelial microparticles (EMPs) and endothelial progenitor cells (EPCs) are considered as a new perspective reflection of the endothelial injury and repair status. Our previous studies have demonstrated that berberine improved endothelial function and arterial stiffness in healthy subjects. In this study, we further investigated the effects of berberine on regulating the circulating EMPs and EPCs, and preventing endothelial dysfunction and arterial stiffness in spontaneously hypertensive rats (SHRs). Methods: Twenty male SHRs were randomly divided into two groups: Berberine-treated SHR group and vehicle-treated SHR group. The SHR rats were intragastrically treated with physiologic saline, berberine 50 mg/kg.d or vehicle for 4 weeks, respectively. Ten male Wistar-Kyoto (WKY) rats treated with vehicle served as normotensive controls. Tail systolic blood pressure was monitored every 2 weeks. At the end of the study, aortic pulse wave velocity (aPWV) was measured in vivo, and aorta were collected for measurement of endothelium-dependent vasodilation and immunohistological staining of elastic fiber. Peripheral blood was collected for circulating EMP detection and EPC culture. Results: Compared to normotensive rats, hypertensive rats displayed significantly higher circulating CD31+/CD42− MPs, lower number and colony-forming units (CFUs) of EPCs, worse endothelium-dependent vasodilation, and faster aPWV. Berberine treatment in SHRs partly reduced the blood pressure and circulating EMPs, and augmented EPC numbers and CFUs. In addition, berberine preserved arterial elasticity by lowering aPWV and increasing the content of arterial media elastin fiber, and improved endothelial function by maintaining better endothelium-dependent vasodilation. Robust relationship was observed among circulating CD31+/CD42− MPs, EPC numbers and aPWV. Conclusions: Abnormal changes of circulating EMPs and EPCs in SHRs are associated with endothelial dysfunction and arterial stiffness. Berberine may be a novel therapeutic option for the hypertension-related vascular injury in SHRs.

Detrimental Impact of Low Birth Weight on Circulating Number and Functional Capacity of Endothelial Progenitor Cells in Healthy Children: Role of Angiogenic Factors

To provide a comprehensive assessment of the relationship of birth weight with both endothelial progenitor cell function and angiogenic factors in children. Anthropometric measures, biochemical profile, endothelial progenitor cell number, endothelial progenitor cell colony-forming units, vascular endothelial growth factor-A, and nitric oxide plasma levels of 58 children aged 7-11 years were determined. A positive correlation was observed between birth weight and circulating endothelial progenitor cell number (r= 0.461; P= .001), endothelial progenitor cell colony-forming units (r= 0.512; P < .001), vascular endothelial growth factor-A (r= 0.407; P= .002), and nitric oxide (r= 0.547; P < .001) levels, whereas the adjustment for prematurity, family history of cardiovascular disease, and systolic blood pressure levels did not modify these associations. Low birth weight was associated with a decrease in the circulating/functional capacity of endothelial progenitor cells among healthy children, independent of traditional cardiovascular risk factors. This detrimental impact was accompanied by lower circulating levels of angiogenic factors.

Human peripheral blood mononuclear cells enriched in endothelial progenitor cells via quality and quantity controlled culture accelerate vascularization and wound healing in a porcine wound model

The transplantation of endothelial progenitor cells (EPCs) is used to promote wound angiogenesis. In patients with chronic wounds and accompanying morbidities, EPCs are often compromised in number and function. To overcome these limitations, we previously developed a quality and quantity controlled (QQ) culture system to enrich peripheral blood mononuclear cells (PBMNCs) in EPCs. To evaluate the wound healing efficacy of mononuclear cells (MNCs) harvested after QQ culture (QQMNCs), preclinical studies were performed on large animals. MNCs harvested from the blood of healthy human subjects were cultured in the presence of angiogenic cytokines and growth factors in a serum-free medium for 7 days. A total of 5 × 106 QQMNCs per full-thickness skin defect or control saline was injected into wounds induced in cyclosporine-immunosuppressed pigs. EPC colony-forming assays revealed a significantly higher number of definitive (partially differentiated) EPC colony-forming units in QQMNCs. Flow cytometry evaluation of QQMNC surface markers showed enrichment of CD34+ and CD133+ stem cell populations, significant reduction in CCR2+ cell percentages, and a greater than 10-fold increase in the percentage of anti-inflammatory M2-type macrophages (CD206+ cells) compared with PBMNCs. Wounds treated with QQMNCs had a significantly higher closure rate. Wounds were harvested, frozen, and sectioned at day 21 postoperatively. Hematoxylin and eosin staining revealed that the epithelization of QQMNC-treated wounds was more advanced than in controls. Treated wounds developed granulation tissue with more mature collagen and larger capillary networks. CD31 and human mitochondrial co-staining confirmed the presence of differentiated human cells within newly formed vessels. Real-time polymerase chain reaction (PCR) showed upregulation of interleukin 6 (IL-6), IL-10, and IL-4 in the wound bed, suggesting paracrine activity of the transplanted QQMNCs. Our data demonstrate for the first time that QQ culture of MNCs obtained from a small amount of peripheral blood yields vasculogenic and therapeutic cells effective in wound healing.

Effect of vitamin D on endothelial progenitor cells function

BackgroundEndothelial progenitor cells (EPCs) are a population of bone marrow-derived cells, which have an important role in the process of endothelialization and vascular repair following injury. Impairment of EPCs, which occurs in patients with diabetes, was shown to be related to endothelial dysfunction, coronary artery disease (CAD) and adverse clinical outcomes. Recent evidence has shown that calcitriol, the active hormone of vitamin D, has a favorable impact on the endothelium and cardiovascular system. There is limited data on the effect of vitamin D on EPCs function.AimTo examine the in vitro effects of Calcitriol on EPCs from healthy subjects and patients with diabetes.MethodsFifty-one patients with type 2 diabetes (60±11 years, 40% women, HbA1C: 9.1±0.8%) and 23 healthy volunteers were recruited. EPCs were isolated and cultured with and without calcitriol. The capacity of the cells to form colony-forming units (CFUs), their viability (measured by MTT assay), KLF-10 levels and angiogenic markers were evaluated after 1 week of culture.ResultsIn diabetic patients, EPC CFUs and cell viability were higher in EPCs exposed to calcitriol vs. EPCs not exposed to calcitriol [EPC CFUs: 1.25 (IQR 1.0–2.0) vs. 0.5 (IQR 0.5–1.9), p < 0.001; MTT:0.62 (IQR 0.44–0.93) vs. 0.52 (IQR 0.31–0.62), p = 0.001]. KLF-10 levels tended to be higher in EPCs exposed to vitamin D, with no differences in angiopoietic markers. In healthy subjects, calcitriol supplementation also resulted in higher cell viability [MTT: 0.23 (IQR 0.11–0.46) vs. 0.19 (0.09–0.39), p = 0.04], but without differences in CFU count or angiopoietic markers.ConclusionIn patients with diabetes mellitus, in vitro vitamin D supplementation improved EPCs capacity to form colonies and viability. Further studies regarding the mechanisms by which vitamin D exerts its effect are required.

Endothelial progenitor cells as a biological marker of peripheral artery disease.

The role of endothelial progenitor cells (EPCs) in peripheral artery disease (PAD) remains unclear. We hypothesized that EPC mobilization and function play a central role in the development of endothelial dysfunction and directly influence the degree of atherosclerotic burden in peripheral artery vessels. The number of circulating EPCs, defined as CD34(+)/KDR(+) cells, were assessed by flow cytometry in 91 subjects classified according to a predefined sample size of 31 non-diabetic PAD patients, 30 diabetic PAD patients, and 30 healthy volunteers. Both PAD groups had undergone endovascular treatment in the past. As a functional parameter, EPC colony-forming units were determined ex vivo. Apart from a broad laboratory analysis, a series of clinical measures using the ankle-brachial index (ABI), flow-mediated dilatation (FMD) and carotid intima-media thickness (cIMT) were investigated. A significant reduction of EPC counts and proliferation indices in both PAD groups compared to healthy subjects were observed. Low EPC number and pathological findings in the clinical assessment were strongly correlated to the group allocation. Multivariate statistical analysis revealed these findings to be independent predictors of disease appearance. Linear regression analysis showed the ABI to be a predictor of circulating EPC number (p=0.02). Moreover, the functionality of EPCs was correlated by linear regression (p=0.017) to cIMT. The influence of diabetes mellitus on EPCs in our study has to be considered marginal in already disease-affected patients. This study demonstrated that EPCs could predict the prevalence and severity of symptomatic PAD, with ABI as the determinant of the state of EPC populations in disease-affected groups.

Proliferation of Peripheral Blood-derived Endothelial Progenitor Cells from Stable Angina Subjects

BACKGROUND: A population of circulating Endothelial Progenitor Cells (EPCs) has been reported to play important role in maintaining endothelial function and integrity. Since EPCs culture is crucial and an optimized medium is currently available. Therefore we conducted a study to investigate whether stable angina subjects peripheral blood-derived EPCs could be cultured in this medium. Here, we performed study to detect EPCs characteristics and extracellular signalregulated kinase (Erk)1/2 Mitogen-Activated Protein Kinase (MAPK) pathway as possible underlying pathway for EPCs proliferation.METHODS: Peripheral blood EPCs from 8 stable angina subjects were cultured in an optimized medium with/without addition of supplement for 1 or 3 days. Then, the membrane of cultured EPCs were detected with immunofluorescence method for CD34, Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) and CD133. Colony forming unit (CFU) enumeration was performed. XTT Cell proliferation assay was performed to assess EPCs growth after 1 and 3-days culture. The western blot analysis was performed to detect possible activation of Erk1/2 MAPK.RESULTS: Number of EPCs and CFU cultured for 3 days were significantly higher than the ones cultured for 1 day (p=0.012). EPCs membrane markers from stable angina subjects were detected as well as CFUs were formed. There were significant increase of EPCs number, CFUs number and phosphorylated-Erk2 amount when the groups with and without supplement were compared (p&lt;0.05). Meanwhile U0126, a MAPK Erk1/2 (MEK1/2) inhibitor, significantly inhibited the supplement-induced EPCs number, CFUs number and phosphorylated-Erk2 amount (p&lt;0.05).CONCLUSION: Our results showed that ERK2 MAPK signaling pathway might play an important role in supplement-induced peripheral blood EPCs proliferation in subjects with stable angina.KEYWORDS: endothelial progenitor cell, EPC, p42, Erk2, proliferation

Endothelial progenitor cells in diabetic patients with myocardial infarction – Can statins improve their function?

The effect of statins on endothelial progenitor cells (EPCs) function derived from diabetic patients (DMpts) with acute myocardial infarction (AMI) is unknown. In this study we assess the response of early and late EPCs from diabetic versus non-diabetic patients (NDMpts) with AMI to statins. EPCs were obtained from 10 diabetic and 10 age-matched non-diabetic male patients with AMI. For each patient, cultures of early and late EPCs were performed under 4 different conditions: normal glucose concentration (control); high glucose concentration; normal glucose concentration with atorvastatin supplementation and normal glucose concentration with pravastatin supplementation. To compare the effect of these treatments on EPC function in DMpts versus NDMpts, we performed in vitro: EPC colony-forming units (CFU) assay; cell cycle analysis; viability assessment and expression of the surface markers CXCR4, CD133, CD34 and KDR. Under control conditions, CFU numbers were reduced in DMpts-derived EPCs when compared to those of NDMpts (1.4±0.8 vs 2.6±1.2 CFU/well, P=0.021). When early EPCs from DMpts were cultured in the presence of statins, CFU capacity was restored, surmounting that of NDMpts under control conditions. Statins significantly improved viability of early EPCs and delayed the onset of late EPCs senescence, even in cells from DMpts. In addition, statins induced approximately a 2-fold increase in the proportion of late EPCs in S-phase of the cell cycle (P<0.05). Statins have a beneficial effect on both early and late EPCs from DMpts with AMI. Despite the functional impairment of EPCs from DMpts, they exhibit similar responsiveness to statins as equivalent cells from NDMpts.

Regular exercise training increases the number of endothelial progenitor cells and decreases homocysteine levels in healthy peripheral blood.

Endothelial progenitor cells (EPCs) are known to play an important role in the repair of damaged blood vessels. We used an endothelial progenitor cell colony-forming assay (EPC-CFA) to determine whether EPC numbers could be increased in healthy individuals through regular exercise training. The number of functional EPCs obtained from human peripheral blood-derived AC133 stem cells was measured after a 28-day regular exercise training program. The number of total endothelial progenitor cell colony-forming units (EPC-CFU) was significantly increased compared to that in the control group (p=0.02, n=5). In addition, we observed a significant decrease in homocysteine levels followed by an increase in the number of EPC-CFUs (p=0.04, n=5), indicating that the 28-day regular exercise training could increase the number of EPC colonies and decrease homocysteine levels. Moreover, an inverse correlation was observed between small-endothelial progenitor cell colony-forming units (small-EPC-CFUs) and plasma homocysteine levels in healthy men (r=-0.8125, p=0.047). We found that regular exercise training could increase the number of EPC-CFUs and decrease homocysteine levels, thus decreasing the cardiovascular disease risk in men.

Plasma levels of stromal cell-derived factor-1 (CXCL12) and circulating endothelial progenitor cells in women with idiopathic heavy menstrual bleeding

Do plasma levels of stromal cell-derived factor-1 (CXCL12, sometimes termed SDF-1) and the numbers of circulating endothelial progenitor cells (EPCs), EPC colony-forming units (EPC-CFU) and mature endothelial cells (ECs) differ between women with idiopathic heavy menstrual bleeding of endometrial origin (HMB-E) and controls and are they related to plasma levels of other angiogenic growth factors? Angiogenesis is altered in women with HMB-E, characterized by a reduction in mean plasma levels of CXCL12, a low number of EPCs-CFUs and a high level of circulating ECs. Plasma levels of CXCL12 are significantly higher during the proliferative than the secretory phase of the menstrual cycle in healthy women and exhibit a negative correlation with blood EPC-CFUs. A prospective cohort study in a university hospital setting. Between 2008 and 2009 10 HMB-E patients were recruited from Karolinska University Hospital. Ten healthy women were also included in the analysis. Ten healthy control women and 10 HMB-E patients, all with regular menstrual cycles, provided 4 blood samples during a single menstrual cycle: 2 in the proliferative phase, 1 at ovulation and 1 in the secretory phase. We assessed plasma levels of CXCL12, vascular endothelial growth factor A(165) (VEGFA), basic fibroblast growth factor (bFGF) and granulocyte and granulocyte-macrophage colony-stimulating factors by ELISA. We counted circulating EPC-CFUs by culture, and ECs and EPCs by flow cytometry and immunostaining for cell surface markers. Plasma levels of CXCL12 were significantly lower in HMB-E patients compared with control women (P < 0.0001), with a significant decrease (P = 0.013) between the proliferative phase and ovulation. VEGFA showed a trend towards the same decreasing pattern as CXCL12, although not statistically significant (P = 0.086), whereas systemic VEGFA levels in control women remained unchanged across the different phases of the menstrual cycle (P = 0.473). HMB-E patients had a lower number of EPC-CFUs compared with control women (P = 0.014), with a positive correlation between the level of CXCL12 and EPC-CFUs (r = 0.428; P = 0.047). Whilst the level of circulating endothelial cells in HMB-E patients was higher than in control women, this did not reach statistical significance. In contrast, the levels of the hematopoietic/EPC marker CD34 were significantly lower in HMB-E patients than control women (P < 0.020). Small sample, unknown source of CXCL12, unknown balance between influx and efflux of EPCs from bone marrow and to the endometrium. Our results indicate that CXCL12 may play an important role in physiological angiogenesis in the endometrium, and that low and dysregulated levels of CXCL12 in women with HMB-E could affect vessel quality, integrity and repair. Financial support was provided through the regional agreement on medical training and clinical research (ALF) between the Stockholm County Council and Karolinska Institutet (number 20110258). This study was also supported by grants from the Swedish Labor Market Insurance. The authors have no conflict of interest to declare.

The dual PPAR-alpha/gamma agonist aleglitazar increases number and function of endothelial progenitor cells: implications for vascular function and atherogenesis

Background: Endothelial progenitor cells (EPC) improve endothelial function and promote vascular repair. Aleglitazar combines lipid-modifying effects of PPAR-α agonists (fibrates) and insulin-sensitizing effects of PPAR-γ agonists (thiazolidinediones). We studied the effects of the novel dual peroxisome proliferator-activated receptor (PPAR)-α/γ agonist aleglitazar on glucose tolerance, EPC, endothelial function, neoangiogenesis and atherosclerosis in mice. Methods and results: C57Bl/6 wild-type (WT, normal chow), ApoE-/- mice (Western-type diet) and eNOS-/- mice were treated with aleglitazar (10 mg/kg/d, i.p.) or vehicle. Aleglitazar enhanced the expression of both the PPAR-α and PPAR-γ target genes and normalized glucose tolerance in cholesterol-fed ApoE-/- mice. In WT mice, aleglitazar treatment upregulated sca-1/VEGFR-2-positive EPC in the blood (153±10%) and bone marrow (197±22%), and upregulated spleen-derived diLDL/lectin-positive EPC (182±8%). Furthermore, aleglitazar augmented EPC migration (186±6% vs. controls) and enhanced neoangiogenesis (vascularized disk area 178±18% vs. controls). The effects of the dual PPAR-α/γ agonist on EPC number and function were abolished in eNOS knock-out mice. In ApoE-/- mice, aleglitazar upregulated EPC number and function, potently improved endothelium-dependent vasodilation and markedly reduced the formation of atherosclerotic plaques (plaque area/total lumen area 2.3±0.8% vs. 10.1±1.9% after 6 weeks and 22±2.2% vs. 36±2.1% after 8 weeks treatment). OilRed staining of hepatic sections showed a profound reduction of liver steatosis in ApoE-/- mice. In cultured human EPC, aleglitazar increased migration and colony forming units in a concentration-dependent manner. Furthermore, oxidative stress-induced EPC apoptosis and protein expression of p53 were reduced, while telomerase activity and expression of phospho-eNOS (S1177) and phospho-Akt were elevated. Comparative and inhibitor experiments revealed that aleglitazar's effects on EPC migration and colony forming units (CFU) were mediated by both PPAR-α and -γ signaling. E.g., aleglitazar treatment (10nM/l) induced EPC migration (304±21% vs. controls) and CFU (300±67% vs. controls) comparably to a co-stimulation of EPC with pioglitazone 10μM/l+fenofibric acid (150μM/l) (Migration: 332±28%; CFU: 289±72%). Conclusions: The dual PPAR-α/γ agonist aleglitazar augments number, function and survival of endothelial progenitor cells in an Akt- and eNOS-dependent fashion. The effects on EPC correlate with improved neoangiogenesis, restored endothelial function and prevention of atherosclerosis.