Decrease In Endothelial Progenitor Cells Research Articles

Intravenous iron administration in patients with heart failure alters endothelial homeostasis with early release of endothelial microvesicles and subsequent mobilization of endothelial progenitor cell

Abstract Background Intravenous iron supplementation is an established therapy for patients with heart failure (HF) with reduced ejection fraction (LV-EF<50%) and iron deficiency, resulting in reduced risk of HF hospitalization. Since iron may induce oxidative stress, inflammation and apoptosis in endothelial cells, concerns persist regarding potential adverse vascular effects of iron therapy. To evaluate endothelial health following ferric carboxymaltose (FCM) administration, we assessed the profile of circulating endothelial microvesicles (EMVs) and endothelial progenitor cells (EPCs) in a cohort of 23 HF patients using flow cytometry. Results Compared to healthy control subjects (HC; n=15), baseline levels of CD31+/CD41- EMVs were significantly higher and EMVs featured a more apoptotic phenotype in HF patients. Following administration of 1000mg FCM, EMV levels showed an early but transient increase (Fig. 1) and displayed an altered phenotype profile with dominant augmentation of EMVs expressing the inducible markers CD62E and CD54, indicating endothelial activation and injury. Levels of circulating vasoregenerative CD45lowCD34+KDR+ EPCs were lower in HF patients and FCM application resulted in an early decrease of EPCs followed by substantial mobilization into the circulation after one week (Fig. 2). Levels of EMVs and EPCs returned to baseline values within two and four weeks, respectively. Compared to HF patients with preserved renal function, the EMV/EPC ratio was higher in HF patients whereas EPC mobilization was diminished with additional chronic kidney disease, suggesting impaired vascular repair capacity. In vitro experiments with cultured endothelial cells revealed that FCM at therapeutic concentrations dose-dependently promotes endothelial apoptosis, increases expression of adhesion molecules and CXCL12, and triggers the generation of EMVs. Conclusion Intravenous iron supplementation with FCM in HF patients provokes a biphasic response with early release of CD62E+ and CD54+ enriched EMVs and subsequent mobilization of EPCs, suggesting temporary aggravation of endothelial dysfunction upon FCM supplementation and consecutive engagement of a vascular defense program.

Deciphering mechanisms of cardiomyocytes and non-cardiomyocyte transformation in myocardial remodeling of permanent atrial fibrillation

IntroductionAtrial fibrillation (AF) is the most prevalent cardiac arrhythmia, and it significantly increases the risk of cardiovascular complications and morbidity, even with appropriate treatment. Tissue remodeling has been a significant topic, while its systematic transcriptional signature remains unclear in AF. ObjectivesOur study aims to systematically investigate the molecular characteristics of AF at the cellular-level. MethodsWe conducted single-nuclei RNA-sequencig (snRNA-seq) analysis using nuclei isolated from the left atrial appendage (LAA) of AF patients and sinus rhythm. Pathological staining was performed to validate the key findings of snRNA-seq. ResultsA total of 30 cell subtypes were identified among 80, 592 nuclei. Within the LAA of AF, we observed a specific subtype of dedifferentiated cardiomyocytes (CMs) characterized by reduced expression of cardiac contractile proteins (TTN and TRDN) and heightened expression of extracellular-matrix related genes (COL1A2 and FBN1). Transcription factor prediction analysis revealed that gene expression patterns in dedifferentiated CMs were primarily regulated by CEBPG and GISLI. Additionally, we identified a distinct subtype of endothelial progenitor cells (EPCs) demonstrating elevated expression of PROM1 and KDR, a population decreased within the LAA of AF. Epicardial adipocytes disclosed a reduced release of the anti-inflammatory and anti-fibrotic factor PRG4, and an augmented secretion of VEGF signals targeting CMs. Additionally, we noted accumulation of M2-like macrophages and CD8+ T cells with high pro-inflammatory score in LAA of AF. Furthermore, the analysis of intercellular communication revealed specific pathways related to AF, such as inflammation, extracellular matrix, and vascular remodeling signals. ConclusionsThis study has discovered the presence of dedifferentiated CMs, a decrease in endothelial progenitor cells, a shift in the secretion profile of adipocytes, and an amplified inflammatory response in AF. These findings could offer crucial insights for future research on AF and serve as valuable references for investigating novel therapeutic approaches for AF.

Intravenous iron supplementation in heart failure patients induces temporary endothelial dysfunction with release of endothelial microvesicles.

Intravenous iron supplementation is an established therapy for patients with heart failure (HF) and concomitant iron deficiency reducing the risk of HF hospitalization. However, concerns persist regarding potential adverse vascular effects, since iron may induce oxidative stress, inflammation, and apoptosis of endothelial cells. To assess endothelial health following ferric carboxymaltose (FCM) administration, we analyzed the profile of circulating endothelial microvesicles (EMVs) and endothelial progenitor cells (EPCs) in a cohort of 23 HF patients using flow cytometry. Compared to healthy subjects, baseline levels of CD31+/CD41- EMVs were higher and EMVs featured a more apoptotic phenotype in HF patients. Following FCM administration, EMV levels showed a rapid but transient increase and displayed an altered phenotype profile with dominant augmentation of EMVs expressing inducible markers CD62E and CD54, indicating endothelial inflammatory activation and injury. Levels of circulating vasoregenerative CD45lowCD34+KDR+ EPCs were lower in HF patients and FCM application resulted in an early decrease of EPCs followed by substantial mobilization into the circulation after one week. Levels of EMVs and EPCs returned to baseline values within two and four weeks, respectively. HF patients with additional chronic kidney disease showed an elevated EMV/EPC ratio and diminished EPC mobilization, suggesting impaired vascular repair capacity. Providing a mechanistic link, in vitro experiments with cultured endothelial cells revealed that FCM dose-dependently promotes endothelial apoptosis, increases expression of adhesion molecules and CXCL12, and triggers generation of EMVs. Intravenous iron supplementation with FCM in HF patients induces a biphasic response with initial increased release of CD62E+ and CD54+ enriched EMVs and subsequent mobilization of EPCs, indicating endothelial dysfunction upon FCM and suggesting consecutive engagement of a defense program aimed to reconstitute vascular health.

The decrease of endothelial progenitor cells caused by high altitude may lead to coronary heart disease.

The purpose of this study was to explore the relationship between the number of endothelial progenitor cells (EPCs) and coronary heart disease (CHD). A total of 24 patients with CHD were chosen from Lanzhou City and Xianyang City, and then, 24 healthy controls who matched the CHD group in gender, age and address were chosen as control group. C-reactive protein (CRP) and c-reaction protein (hs-CRP) were detected. The levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), homocysteine (Hcy), hypoxia-inducible factor-1 (HIF-1α) and stromal cell-derived factor 1 (SDF-1α) were detected. The number of EPCs in control groups was both increased compared with CHD group (p<0.05). The number of EPCs in Xianyang control group was increased compared with Lanzhou control group (p<0.05). Compared with the control group, the levels of TC, LDL and CRP in the CHD group were higher (p<0.05). Compared with Lanzhou control group, Hcy level was decreased in Lanzhou CHD group (p<0.05). Compared with Xianyang control group, the levels of IL-8 and VEGF were increased, but the levels of HIF-1α and Hcy were decreased in the Xianyang CHD group (p<0.05). The expressions of IL-8, VEGF, Hcy and HIF-1α were increased in Lanzhou control group than the Xianyang control group (p<0.05). In Lanzhou CHD group, Spearman correlation analysis showed that the number of EPCs was negatively related to hs-CRP content (r=-0.631, p<0.05). The decrease of EPCs caused by high altitude may increase the expressions of various cytokines, leading to the occurrence of CHD.

Increased circulating endothelial progenitor cells (EPCs) in prepubertal children born prematurely: a possible link between prematurity and cardiovascular risk.

Endothelial progenitor cells (EPCs) ensure vascular integrity and neovascularization. No studies have investigated EPCs in preterm-born children beyond infancy. One hundred and thirty-six prepubertal children were enrolled: 63 preterm and 73 born at term (controls). Circulating CD34(+)/VEGFR-2(+)/CD45(-) and CD34(+)/VEGFR-2(+)/CD45dim EPCs were measured in preterm-born children compared to controls. Body mass index (BMI), waist-to-hip ratio (WHR), neck circumference, systolic and diastolic blood pressure (SBP and DBP, respectively), fasting glucose, insulin, lipid profile, common carotid and abdominal aortic intima-media thickness (cIMT and aIMT, respectively), endothelium-dependent brachial artery flow-mediated dilation (FMD), and echocardiographic parameters were also assessed. Circulating CD34(+)/VEGFR-2(+)/CD45(-) and CD34(+)/VEGFR-2(+)/CD45dim EPCs were significantly higher in preterm-born children compared to controls (p < 0.001 and p < 0.001, respectively). In total study population and in the preterm-born group, EPCs were significantly lower in children born to mothers with gestational diabetes compared to non-diabetic mothers. Prematurity was associated with higher WHR, neck circumference, SBP, DBP, cIMT, aIMT, mean pressure, and velocity of pulmonary artery; the peak velocity of the brachial artery was significantly lower in children born prematurely. In multiple regression analysis, preterm birth and maternal gestational diabetes were recognized as independent predictors of EPCs. Circulating EPCs were increased in prepubertal preterm-born children in comparison with peers born full-term. Maternal gestational diabetes was associated with a decrease in EPCs. Mounting evidence supports the adverse effect of prematurity on cardiovascular health. However, the underlying mechanisms that could lead to endothelial dysfunction in preterm-born individuals are not fully understood. Endothelial progenitor cells (EPCs) ensure vascular integrity, normal endothelial function and neovascularization. No studies have investigated the EPCs counts in peripheral blood beyond infancy in children born prematurely. Circulating EPCs were significantly higher in preterm-born prepubertal children compared to controls, thus indicating that prematurity is possibly associated with endothelial damage. In total study population and in the preterm-born group, maternal gestational diabetes was associated with decreased EPCs concentrations.

Abstract P1-10-02: A phase II study of copper-depletion using tetrathiomolybdate in patients with breast cancer at high risk for recurrence: Updated results

Abstract Background: Metals have emerged as a viable therapeutic target for a new generation of anti-cancer and anti-metastatic agents. Copper, an essential trace element, serves as an important catalytic cofactor in several biological functions and has emerged as an essential factor in carcinogenesis. Among other elements, bone marrow derived VEGFR2+ endothelial progenitor cells (EPCs) and copper-dependent lysyl oxidase (LOX) are key elements in tumor progression. We hypothesized tetrathiomolybdate (TM)-associated copper depletion (CD) inhibits tumor metastases by reducing the number of EPCs and other copper dependent processes in the pre-metastatic niche. These results are an update of our previously reported study (Chan N, Willis A, Kornhauser N et al. Influencing the Tumor Microenvironment: Phase 2 Study of Copper Depletion with Tetrathiomolybdate in High Risk Breast Cancer and Preclinical Models of Lung Metastases. Clin Cancer Res. October 21, 2016) with longer follow-up. Methods: A single arm phase II study of breast cancer (BC) patients (pts) at high risk for recurrence, defined as node+ triple negative (TNBC), stage 3 and 4 with no evidence of disease (NED) were enrolled on a trial of CD with TM. TM was given to maintain ceruloplasmin (Cp) levels between 8-16 mg/dl for two years with an extension phase or until relapse. The primary endpoint was a change in EPCs measured by flow cytometry before and during treatment. Secondary endpoints included tolerability, safety, PFS and LOXL-2 levels. Results: Seventy-five pts received 2778 cycles of TM on the primary and extension study. The primary study treatment duration was 24 cycles (each cycle is 28 days) plus an extension phase. The median age is 51 years (range 29-66). Forty-five pts have stage 2/3 BC and 30 with stage 4 NED. Forty-eight percent of pts are TNBC and 40% of pts are stage 4 NED. Median Cp levels were monitored with each cycle. A decrease from 28 to 16 (p&amp;lt;0.0001) was seen after one cycle. Interestingly, TNBC pts seemed to have a greater decrease from 23.5 to 13 after one cycle. TM was well tolerated with grade 3/4 toxicities including: reversible neutropenia (2.3%), febrile neutropenia (0.04%), fatigue (0.2%). Five-year analysis showed a decrease in EPC's (p=0.004) and LOXL-2 (p&amp;lt;0.001). At a median follow-up of 7.1 years, the EFS for 75 pts is 71.4%. The EFS for 36 pts with TNBC is 71.7%. EFS for stage 2/3 TNBC is 83% and for stage IV TNBC is 59.3%. Conclusions: TM is safe, well tolerated and appears to affect multiple components of the tumor microenvironment that have been identified in pre-clinical models as important for progression. Ongoing studies in banked specimens are underway to further delineate its effect on copper dependent processes necessary for metastases. Randomized trials are warranted, especially in patients who are at high risk for relapse such as those with TNBC. Citation Format: Sahota S, Willis A, Kornhauser N, Ward M, Cobham M, Cigler T, Moore A, Andreopoulou E, Fitzpatrick V, Schneider S, Prima N, Wiener A, Ko D, De Laurentiis A, Warren JD, Rubinchik A, Mittal V, Vahdat LT. A phase II study of copper-depletion using tetrathiomolybdate in patients with breast cancer at high risk for recurrence: Updated results [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-02.

A phase II study of copper-depletion using tetrathiomolybdate (TM) in patients (pts) with breast cancer (BC) at high risk for recurrence: Updated results.

2557 Background: The tumor microenvironment (TME) plays a critical role in the spread of tumors. Bone marrow derived VEGFR2+endothelial progenitor cells (EPCs) and copper-dependent lysyl oxidase (LOX) are key in tumor progression. We hypothesized TM-associated copper depletion inhibits tumor metastases by reducing the number of EPCs and other copper dependent (CD) processes in the pre-metastatic niche. These results are an update with longer follow-up. Methods: Phase II study of BC pts at high risk for recurrence, defined as node+ triple negative (TNBC), stage 3 and 4 with no evidence of disease (NED) were enrolled on a trial of CD with TM. Ceruloplasmin (Cp) levels were maintained between 8-16 mg/dl for two years with an extension phase or until relapse. The primary endpoint was change in EPCs measured by flow cytometry before and during treatment. Secondary endpoints included tolerability, safety, PFS and LOXL-2 levels. Results: 75 pts received 2650 cycles of TM on primary and extension study. The median age is 51 years (range 29-66). Forty-five pts have stage 2/3 BC and 30 with stage 4 NED. TNBC pts were 48% and 40% of pts are stage 4 NED. Median Cp level decreased from 28 to 16 (p &lt; 0.0001) after one cycle. Copper depletion was most efficient in TNBC where Cp levels dropped from 23.5 to 13 after one cycle. TM was well tolerated with grade 3/4 toxicities including: reversible neutropenia (2.3%), febrile neutropenia (0.04%), fatigue (0.2%). Five-year analysis showed a decrease in EPC’s (p = 0.004) and LOXL-2 (p &lt; 0.001). At a median follow-up of 6.9 years, the EFS for 75 pts is 75.6%. PFS for 36 pts with TNBC is 79.2%. EFS for stage 2/3 TNBC is 90% and for stage IV TNBC is 66.7%. Conclusions: TM is safe, well tolerated and appears to affect multiple components of the TME creating an inhospitable environment for tumor progression especially in high risk patients such as TNBC. Randomized trials are warranted, especially in patients at high risk for relapse. Clinical trial information: UL1TR000457.

Abstract LB-349: Copper depletion as a strategy to affect the tumor microenvironment in breast cancer patients at high risk of relapse and in triple negative preclinical models of breast cancer: Updated results of a phase II study of tetrathiomolybdate (TM) in breast cancer (BC) patients (pts) at high risk for recurrence

Abstract Background: The pre-metastatic niche mobilizes copper to assist in conditioning the tumor microenvironment facilitating tumor progression. This includes bone marrow derived VEGFR2+ endothelial progenitor cells (EPCs) and copper-dependent lysyl oxidase (LOX). We hypothesized that TM-associated CD would reduce EPCs and other copper dependent processes in the pre-metastatic niche in BC pts. Methods: Stage 2 triple negative BC, Stage 3 and stage 4 without any evidence of disease, (NED) BC pts, received oral TM to maintain ceruloplasmin (Cp) between 8-17mg/dL for 2 years or until relapse. Endpoints included change in EPCs, safety, disease-free (DFS) and overall survival (OS) and effect on biomarkers. Laboratory studies: MDA-LM2-luciferase cells were implanted into CB17-SCID mice, and treated with TM or water. Tumor progression was quantified by bioluminescence imaging (BLI), CD status by Cp oxidase levels, lysyl oxidase (LOXL2) activity by ELISA, and collagen deposition by Picrosirius Red staining and Second Harmonic Generation (SHG) imaging analysis. Results: 75 pts (45 stage 2/3 BC, 30 stage 4 NED) received over 2400 cycles of TM on the primary and extension studies. Median age: 51 yrs (range 29-66). Of the 75 pts, 48% had TNBC, 40% were stage 4 NED. Median Cp level decreased from 28 at baseline to 15.5 (p&amp;lt;0.0001) after one cycle. Grade 3/4 toxicities included reversible neutropenia (2.3%), anemia (0.04%), leukopenia (1.2%) and fatigue (0.09%). CD was associated with a decrease in EPCs (p = 0.0014) and serum LOX (p&amp;lt;0.001). At a median follow-up of 5.9 years, the PFS for all 75 pts is 72%, including a PFS of 90% for all stage 2/3 pts with TNBC. The overall survival is 84%. Relapse after two years is a rare event. In pre-clinical models, TM had no effects on the primary tumor, but decreased secondary lung metastases (p = 0.04) as seen by BLI. Western blots demonstrated decreased LOX in the pre-metastatic lungs vs controls (p = 0.03), and IHC showed reduced collagen cross-linking by image J (p = 0.01). SHG analysis showed reduced collagen fiber length. Conclusions: TM is safe, well tolerated and appears to affect multiple copper dependent biologic processes in the tumor microenvironment known to be important for tumor progression, most striking in TNBC. Randomized trials in a high risk for relapse population are warranted. Citation Format: Eleni Nackos, Sharrell Lee, Amy Willis, Naomi Kornhauser, Maureen Ward, Marta Cobham, Tessa Cigler, Anne Moore, Veronica Fitzpatrick, Sarah Schneider, Alysia Wiener, Jessica Guillaume-Abraham, Bo Ri Seo, J David Warren, Anna Rubinchik, Claudia Fischbach, Vivek Mittal, Linda Vahdat. Copper depletion as a strategy to affect the tumor microenvironment in breast cancer patients at high risk of relapse and in triple negative preclinical models of breast cancer: Updated results of a phase II study of tetrathiomolybdate (TM) in breast cancer (BC) patients (pts) at high risk for recurrence. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-349.

Abstract P3-02-02: Targeting the tumor microenvironment: A phase II study of copper-depletion using tetrathiomolybdate (TM) in patients (pts) with breast cancer (BC) at high risk for recurrence

Abstract Background: Bone marrow derived VEGFR2+ endothelial progenitor cells (EPCs) and copper-dependent pathways, including lysyl oxidase (LOX), are critical components to remodeling the tumor microenvironment and establishing the pre-metastatic niche. In preclinical models, it has been well established that copper depletion (CD) inhibits tumor progression. We hypothesized that TM-associated CD would reduce EPCs and other copper dependent processes in the pre-metastatic niche in BC pts at high risk for relapse. We investigated the relationship between CD and its effect on EPCs and other components of the tumor microenvironment including LOX, an enzyme critical for cross-linkage of collagen and priming the pre-metastatic niche. Methods: In this single arm, phase II study, BC pts at high risk for recurrence, defined as node+ triple negative (TN), stage 3 and 4 with no evidence of disease (NED) were enrolled on a trial of CD with TM. Pts received oral TM to maintain ceruloplasmin (Cp) between 5-17 mg/dl for 2 years on the primary study. The primary endpoint was change in EPCs measured by flow cytometry before and during treatment with TM. Secondary endpoints included tolerability, safety and effect of copper depletion on other markers including LOX, quantified by ELISA. Results: We enrolled 75 pts. The study treatment duration was 24 cycles (each cycle is 28 days). Over 2200 cycles have been administered. The median age is 51 (range 29-66). 45 pts have Stage 2/3 BC and 30 are Stage 4 NED. TNBC pts represent 48%, and 40% of pts are Stage 4 NED. Median Cp level decreased from 28 at baseline to 15.5 (p&amp;lt;0.0001) after one cycle. Copper depletion was most efficient in TNBC, with 91% achieving a target CP within 4 weeks. TM was well tolerated and the only grade 3/4 toxicities were reversible neutropenia (3.2%) and anemia (0.0005%). CD was associated with a significant decrease in EPCs (p=0.0014) and LOX (p&amp;lt;0.001). At a median follow-up of 5.4 years, the PFS for all 75 pts from the start of TM treatment was 71%, including a PFS of 90% for all stage 2/3 pts with TNBC. The overall survival of all patients enrolled in the trial is 86%. Relapse after two years is a rare event. Conclusions: TM is safe, well tolerated and appears to affect multiple copper dependent biologic processes in the tumor microenvironment known to be important for tumor progression. This seems to be most striking in TNBC. We believe, further phase III trials in a high risk for relapse population are warranted. Citation Format: Nackos E, Willis A, Kornhauser N, Ward M, Andreopoulou E, Cigler T, Moore A, Fitzpatrick V, Cobham M, Schneider S, Wiener A, Guillaume-Abraham J, Warren JD, Rubinchik A, Lane M, Mittal V, Vahdat L. Targeting the tumor microenvironment: A phase II study of copper-depletion using tetrathiomolybdate (TM) in patients (pts) with breast cancer (BC) at high risk for recurrence. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-02-02.

Maintenance chemotherapy in children with ALL exerts metronomic-like thrombospondin-1 associated anti-endothelial effect.

Maintenance chemotherapy is an important part of the treatment of ALL in children. It relies on the long-term oral administration of daily low-dose mercaptopurin and weekly low-dose methotrexate. Although it has been used in the clinic for decades, its mechanisms of action remain unclear. Here, we investigated different angiogenic and immune biomarkers to gain insights into the mechanisms of action of maintenance therapy in children with ALL. We thus monitored circulating endothelial cells (CEC), endothelial progenitor cells (EPC) and endothelial microparticles (EMP), pro-angiogenic factors (VEGF, VEGFR-1 and Ang-2), anti-angiogenic factor thrombospondin-1 (THBS1) and regulatory T lymphocytes (Treg) in 47 children with ALL during the maintenance phase of their treatment (at treatment initiation and after 6, 12 and 18 months). We observed a statistically significant decrease in EPC and EMP counts throughout the maintenance phase associated with a significant increase in THBS1 levels. No significant change was detected in other angiogenic markers or in Treg numbers.The results presented here indicate that maintenance therapy in children with ALL exerts its antitumor activity at least in part through anti-angiogenic effects, similar to those induced by metronomic chemotherapy. Larger studies are now warranted to validate these findings and determine their clinical implications.

Endothelial progenitor cells lowering effect and compensative mechanism in electrical burn injury models of rat

This study aimed to investigate the effects of electrical injury on vascular damage and on matrix metalloproteinase (MMPs) levels. Thirty male Wistar albino rats were divided into five groups (n = 6), including a control group (untreated group), a 600 mv electrical burn injury group, a 900 mv electrical burn injury group, a 1200 mv electrical burn injury group, and a 1500 mv electrical burn injury group. Endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) were analyzed using flow cytometry. The levels of MMP-9, MMP-3, nitric oxide (NO), vascular endothelial growth factor (VEGF), and vascular cell adhesion molecule (VCAM) were assayed using an enzyme linked immunosorbent assay. Levels of EPCs were significantly lower in all electrical burn injury groups compared with the control group (p < 0.05). The levels of NO and VEGF were significantly increased in all electrical burn injury groups compared with the control group (p < 0.05). The levels of MMP-9 were significantly higher in the 1200 mv electrical burn injury group compared with the control group or the 900 mv electrical burn injury group (p < 0.05). There were no significant differences in MMP-3, VCAM, and CECs levels between groups (p > 0.05). This electrical burn injury model shows a significant decrease in endothelial progenitor cells and an increase in VEGF, NO, and MMP-9 as the compensating mechanism.

Altered chemokine signalling in endothelial progenitor cells from acute ulcerative colitis patients.

Ulcerative colitis (UC) is a chronic, idiopathic, inflammatory bowel disease, characterized by alternating stages of clinically active and inactive disease. UC exhibits several inflammatory characteristics, including immune activation, leukocyte infiltration, and altered vascular density. In UC, many of the upregulated inflammatory cytokines are proangiogenic and are released by diverse cell populations, such as infiltrating immune cells and endothelial cells (EC). Increasing evidences suggest that neovascularisation may involve also endothelial progenitor cells (EPCs). In this study we evaluated EPCs recruitment and homing, assessed by CXCR4 expression, in both acute and remitting phase of UC. We report an overall decrease of EPCs in UC patients (controls = 97,94 ± 37,34 cells/mL; acute = 31,10 ± 25,38 cells/mL; remitting = 30,33 ± 19,02 cells/mL; P < 0.001 for both UC groups versus controls). Moreover CXCR4+-EPCs, committed to home in inflammatory conditions, were found to be reduced in acute UC patients compared to both remitting patients and controls (acute = 3,13 ± 4,61 cells/mL; controls = 20,12 ± 14,0; remitting = 19,47 ± 12,83; P < 0,001). Interestingly, we found that administration of anti-inflammatory drugs in acute UC is associated with an increase in circulating EPCs, suggesting that this therapy may exert a strong influence on the progenitor cells response to inflammatory processes.

Adherence to lifestyle modifications after a cardiac rehabilitation program and endothelial progenitor cells

An increase of endothelial progenitor cells (EPCs) among acute myocardial infarction (AMI) patients participating in a cardiac rehabilitation (CR) program has been reported, but no data on the impact of adherence to lifestyle recommendations provided during a CR program on EPCs are available. It was our aim to investigate the effect of adherence to lifestyle recommendations on EPCs, inflammatory and functional parameters after six months of a CR program in AMI patients. In 110 AMI patients (90 male/20 female; mean age 57.9 ± 9.4 years) EPCs, high sensitivity C-reactive protein (hsCRP), N-terminal pro-brain natriuretic peptide (NT-ProBNP) levels, and cardiopulmonary testings were determined at the end of the CR (T1) and at a six-month follow-up (T2). At T2 we administered a questionnaire assessing dietary habits and physical activity. At T2, we observed a decrease of EPCs (p<0.05), of hsCRP (p=0.009) and of NT-ProBNP (p<0.0001). Patient population was divided into three categories by Healthy Lifestyle (HL) score (none/low, moderate and high adherence to lifestyle recommendations). We observed a significant association between adherence to lifestyle recommendations, increase in EPCs and exercise capacity between T1 and T2 (Δ EPCs p for trend <0.05; ΔWatt max p for trend=0.004). In a multivariate logistic regression analyses, being in the highest tertile of HL score affected the likelihood of an increase of EPC levels at T2 [OR (95% confidence interval): 3.36 (1.0-10.72) p=0.04]. In conclusion, adherence to lifestyle recommendations provided during a CR program positively influences EPC levels and exercise capacity.

Altered Inflammatory Response Is Associated With an Impaired Autonomic Input to the Bone Marrow in the Spontaneously Hypertensive Rat

Autonomic nervous system dysfunction, exaggerated inflammation, and impaired vascular repair are all hallmarks of hypertension. Considering that bone marrow (BM) is a major source of the inflammatory cells (ICs) and endothelial progenitor cells (EPCs), we hypothesized that impaired BM-autonomic nervous system interaction contributes to dysfunctional BM activity in hypertension. In the spontaneously hypertensive rat (SHR), we observed a >30% increase in BM and blood ICs (CD4.8(+)) and a >50% decrease in EPCs (CD90(+).CD4.5.8(-)) when compared with the normotensive Wistar-Kyoto rat. Increased tyrosine hydroxylase (70%) and norepinephrine (160%) and decreased choline acetyl transferase (30%) and acetylcholine esterase (55%) indicated imbalanced autonomic nervous system in SHR BM. In Wistar-Kyoto rat, night time-associated elevation in sympathetic nerve activity (50%) and BM norepinephrine (41%) was associated with increased ICs (50%) and decreased EPCs (350%) although BM sympathetic denervation decreased ICs (25%) and increased EPCs (40%). In contrast, these effects were blunted in SHR, possibly because of chronic downregulation of BM adrenergic receptor α2a (by 50%-80%) and β2 (30%-45%). Application of norepinephrine resulted in increased BM IC activation/release, which was prevented by preadministration of acetylcholine. Electrophysiological recordings of femoral sympathetic nerve activity showed a more robust femoral sympathetic nerve activity in SHR when compared with Wistar-Kyoto rat, peaking earlier in the respiratory cycle, indicative of increased sympathetic tone. Finally, manganese-enhanced MRI demonstrated that presympathetic neuronal activation in SHR was associated with an accelerated retrograde transport of the green fluorescent protein-labeled pseudorabies virus from the BM. These observations demonstrate that a dysfunctional BM autonomic nervous system is associated with imbalanced EPCs and ICs in hypertension.

Reduced circulating endothelial progenitor cells in thromboangiitis obliterans (Buerger’s disease)

To determine the role of endothelial progenitor cells (EPCs) in the pathogenesis of thromboangiitis obliterans (TAO), EPC numbers and colony-forming units, migratory function and tubular structure formation in vitro were compared between 13 young male TAO patients and two age-matched healthy control groups: 11 smokers and 12 non-smokers. TAO patients had significantly lower numbers of EPCs and EPC colonies compared to both non-smokers [190 (97.0-229) vs 528 (380-556), p < 0.001 for EPCs and 0.80 (0.53-1.00) vs 2.80 (2.08-4.00) per mm(2), p = 0.001 for EPC colonies] and smokers [190 (97.0-229) vs 272 (229-326), p = 0.012 for EPCs and 0.80 (0.53-1.00) vs 2.80 (1.80-3.93) per mm(2), p = 0.001 for EPC colonies]. However, there were no significant differences in migratory function or tube formation between the three groups. These results suggest that TAO patients have an intrinsic decrease in EPCs not entirely associated with smoking, which may be the cause of endothelial dysfunction seen in TAO patients leading to the development of this disease at early ages.

Circulating Endothelial Progenitor Cells Decrease in Infants with Bronchopulmonary Dysplasia and Increase after Inhaled Nitric Oxide

BackgroundImpairment of endothelial progenitor cells (EPCs) has been shown to contribute to the development of bronchopulmonary dysplasia (BPD). In the current study, the relationship between EPC changes of after birth and the development of BPD was investigated, and the effects of inhaled nitric oxide (iNO) on EPCs were evaluated.MethodsSixty infants with a gestational age of less than 32 weeks and a birth weight of less than 1500 g were studied. NO was administered to infants who were receiving mechanical ventilation or CPAP for at least 2 days between the ages of 7 and 21 days. EPC level was determined by flow cytometry at birth, 7, 21 and 28 days of age and 36 weeks’ postmenstrual age (PMA), before and after the iNO treatment. Plasma concentrations of vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 and granulocyte-macrophage colony-stimulating factor were determined via immunochemical assay.ResultsTwenty-five neonates developed BPD, 35 neonates survived and did not develop BPD. EPC level was decreased on day 7 and 21 in infants who later developed BPD compared with infants that did not develop BPD. From birth to 21 days of age, BPD infants had a persistently lower VEGF concentration compared with non-BPD infants. No difference was found between the two groups at day 28 or 36 weeks PMA. In infants that later developed BPD, iNO raised the KDR+CD133+ and CD34+KDR+CD133+ EPC numbers along with increasing the level of plasma VEGF.ConclusionEPC level was reduced at 7 days of age in infants with BPD, and iNO increased the EPC number along with increasing the level of VEGF. Further studies are needed to elucidate the mechanism leading to the decrease of EPCs in infants with BPD and to investigate the role of iNO treatment in the prevention of BPD.

Abstract 181: Brain Angiotensin II Elevates Bone Marrow Sympathetic Drive

Introduction: Bone marrow (BM)-derived endothelial progenitor cells (EPCs) contribute to the repair of vasculature, whereas the inflammatory cells (ICs) contribute to the vascular damage. Recently, dysfunctional brain-BM communication has been associated with an impaired BM activity, reflecting a decrease in EPCs and increase in ICs. We hypothesized that an overactive brain RAS leads to elevated sympathetic drive to the BM, which precedes the increase in blood pressure (BP), initiates the EPC/IC imbalance, and contributes to the development of vascular pathophysiology. Methods: Sprague-Dawley (SD) rats were infused with 15ng/kg/min Ang II ICV for 7 days. Following this, indirect BP measurements were taken, and the in situ decerebrated artificially-perfused preparation (DAPR) was used to compare thoracic (tSNA) and femoral BM sympathetic nerve activity (fSNA) between control and Ang II rats. In order to characterize tSNA and fSNA, we monitored the effects of elevated CO 2 (9%) on the phrenic nerve activity (PNA), tSNA and fSNA. PNA-triggered averaging of integrated tSNA and fSNA signals was performed across 100 phrenic cycles, allowing for quantification of averaged tSNA and fSNA during discrete phases of the respiratory cycle: inspiration (I), post-inspiration (P-I), mid-expiration (M-E) and late expiration (L-E). Thus, the peak levels of tSNA and fSNA during each respiratory phase could be compared across preparations. Results: We observed no differences in the BPs of control (95±5 mm Hg) and Ang II rats (96±2 mm Hg). Electrophysiology revealed a characteristic PNA pattern, and peak activity of tSNA and fSNA during the P-I phase, typical of the respiratory-sympathetic coupling. Exposure to 9% CO 2 elevated both the tSNA and fSNA in the control and Ang II rats. However, a 75% increase in the tSNA peak firing, and a 20% increase in the fSNA peak firing were observed in the Ang II rats when compared to the control. Conclusion: These observations demonstrate, for the first time, that elevated brain Ang II, at sub-pressor conditions, results in an increased sympathetic drive to the BM. This may initiate early impaired BM activity, reflecting in an imbalance in BM EPCs and ICs, which may contribute to the development of hypertension-related pathophysiology.

Endothelial progenitor cells are related to glycemic control in children with type 1 diabetes over time.

OBJECTIVEThe risk of cardiovascular death before the age of 40 is 20-fold higher in patients with type 1 diabetes mellitus (T1DM). Endothelial progenitor cells (EPCs) predict cardiovascular morbidity and mortality in patients without diabetes. We hypothesized that EPCs are modified in children with T1DM and are related to characteristics of T1DM such as glycemic control.RESEARCH DESIGN AND METHODSChildren (n = 190; 156 T1DM subjects and 34 control subjects) were included in an observational cohort study and matched for age and sex. EPCs were enumerated by flow cytometry at the beginning (cross-sectional) and 1 year later (longitudinal). To analyze changes of variables during the observation, Δ values were calculated.RESULTSEPCs were significantly reduced in T1DM children versus control subjects (609 ± 359 vs. 1,165 ± 484, P < 0.001). Multivariate regression modeling revealed that glycated hemoglobin A1c (HbA1c) was the strongest independent predictor of EPCs (β = −0.355, P < 0.001). Overall glycemic control at the beginning and end of study did not differ (7.8 ± 1.2 vs. 7.8 ± 1.2 relative %, P = NS), but we observed individual HbA1c changes of −4.30/+3.10 relative %. The strongest EPC increase was observed in the patients with the most favorable HbA1c lowering during the 1-year follow-up. Accordingly, the strongest EPC decrease was demonstrated in the patients with the strongest HbA1c worsening during the time period.CONCLUSIONSThis is the first prospective study demonstrating diminished EPCs in children with T1DM. The association of better glycemic control with an increase in EPC numbers within 1 year suggests that a reduction of the high cardiovascular disease burden might be mediated likewise.

Decrease in endothelial progenitor cells associated with inflammation, but not with endothelial dysfunction in chronic hemodialysis patients

Endothelial progenitor cells (EPC), bone marrow derived cells, are considered to have a pivotal role in maintaining the integrity and repair of the endothelium. Endothelial dysfunction, atherosclerosis and inflammation are implicated for increased CV mortality in uremia. In this study, we aimed to investigate the possible association of EPC with inflammation, endothelial dysfunction and atherosclerosis in chronic hemodialysis (HD) patients. 67 HD patients (male/female: 30/37, mean age: 58 ± 15 years) and 22 healthy controls (male/female: 13/9; mean age: 48 ± 8 years) were included. EPC were cultivated in the fibronectin-covered culture dishes and counted. Also EPC markers were studied by flow cytometry using anti-CD34, anti-CD133 and anti-vascular endothelial growth factor receptor 2 (VEGFR-2) antibodies. Serum levels of IL-6, TNF-α, intercellular cell adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM) and asymmetric dimethyl-arginine (ADMA) were measured by ELISA method. Endothelial function was investigated by measuring flow-mediated dilatation (FMD) of the brachial artery. Carotid intima-media thickness (CIMT) and ratio (CIMR) were also examined. EPC number was decreased in HD patients when compared to controls (63.7 ± 8.9 vs. 101.5 ± 19.6/ high power field, p < 0.001). Also CD34+ cell count was significantly lower in the HD group (2.26 ± 3.52 vs. 6.03 ± 4.73%, p < 0.0001). EPC number was significantly inversely correlated with serum TNF-α levels in HD patients(r: -0.453, p < 0.001) and also in the control group (r = -0.509, p = 0.044). There was an inverse association between VEGFR-2+/CD34+cell count and serum IL-6 levels (r: -0.364, p = 0.006) in HD patients. However, EPC count was not related to FMD and CIMT/CIMR. In HD patients, there was a positive correlation between serum IL-6 levels with CIMT (r = 0.358, p = 0.01) and CIMR was positively correlated with serum ICAM (r = 0.430, p = 0.002). EPC number was decreased in uremia and was associated with inflammation. TNF-α might have specific inhibitory actions on EPC in both HD patients and healthy controls. No relationship was present between EPC and endothelial dysfunction/atherosclerosis.

Evidence of Bacteroides fragilis Protection from Bartonella henselae-Induced Damage

Bartonella henselae is able to internalize endothelial progenitor cells (EPCs), which are resistant to the infection of other common pathogens. Bacteroides fragilis is a gram-negative anaerobe belonging to the gut microflora. It protects from experimental colitis induced by Helicobacter hepaticus through the polysaccharide A (PSA). The aim of our study was to establish: 1) whether B. fragilis colonization could protect from B. henselae infection; if this event may have beneficial effects on EPCs, vascular system and tissues. Our in vitro results establish for the first time that B. fragilis can internalize EPCs and competes with B. henselae during coinfection. We observed a marked activation of the inflammatory response by Real-time PCR and ELISA in coinfected cells compared to B. henselae-infected cells (63 vs 23 up-regulated genes), and after EPCs infection with mutant B. fragilis ΔPSA (≅90% up-regulated genes) compared to B. fragilis. Interestingly, in a mouse model of coinfection, morphological and ultrastructural analyses by hematoxylin-eosin staining and electron microscopy on murine tissues revealed that damages induced by B. henselae can be prevented in the coinfection with B. fragilis but not with its mutant B. fragilis ΔPSA. Moreover, immunohistochemistry analysis with anti-Bartonella showed that the number of positive cells per field decreased of at least 50% in the liver (20±4 vs 50±8), aorta (5±1 vs 10±2) and spleen (25±3 vs 40±6) sections of mice coinfected compared to mice infected only with B. henselae. This decrease was less evident in the coinfection with ΔPSA strain (35±6 in the liver, 5±1 in the aorta and 30±5 in the spleen). Finally, B. fragilis colonization was also able to restore the EPC decrease observed in mice infected with B. henselae (0.65 vs 0.06 media). Thus, our data establish that B. fragilis colonization is able to prevent B. henselae damages through PSA.