Subclinical Thyrotoxicosis and Cardiovascular Risk: Assessment of Circulating Endothelial Progenitor Cells, Proangiogenic Cells, and Endothelial Function.

Background: Subclinical thyrotoxicosis (SCT) is defined by low or undetectable thyroid-stimulating hormones and normal thyroid hormones. The treatment of SCT is uncertain despite being associated with increased cardiovascular risk (CVR) and mortality. Circulating endothelial progenitor cells (cEPCs) and circulating angiogenic cells (CACs) have been found to be reduced in conditions with CVR. We aimed to evaluate whether endothelial function and cEPC and CAC counts were reduced in SCT and to study the in vitro effect of triiodothyronine (T3) on proangiogenic cell (PAC) function from young healthy controls. Methods: cEPCs (quantified by flow cytometry, 20 SCT/20 controls), CACs following in vitro cultures (15 SCT/14 controls), paracrine function of CACs, endothelial function by flow-mediated dilation (FMD, 9 SCT/9 controls), and the effect of T3 on apoptosis and endothelial nitric oxide synthase (eNOS) expression in PACs were studied. Results: p < 0.001, CD133+/VEGFR-2+ 0.4 (0.0-0.7) vs. 0.6 (0.0-4.6), p = 0.009, CD34+/VEGFR-2+ 0.3 (0.0-1.0) vs. 0.7 (0.1-4.9), p = 0.002; while CAC count was similar. SCT predicted a lower cEPC count after adjustment for conventional CVR factors. FMD was lower in SCT subjects versus controls (% mean ± SD, 2.7 ± 2.3 vs. 6.1 ± 2.3, p = 0.005). In vitro studies showed T3 increased early apoptosis and reduced eNOS expression in PACs. Conclusions: In conclusion, SCT is associated with reduced cEPC count and FMD, confirming increased CVR in SCT. Future outcome trials are required to examine if treatment of this subclinical hyperactive state improves cardiovascular outcome. Clinical Trial Registration: http://www.controlled-trials.com/isrctn/, identifier ISRCTN70334066.

The impact of patient sex on the response to intramyocardial mesenchymal stem cell administration in patients with non-ischaemic dilated cardiomyopathy.

AIMS: Sex differences impact the occurrence, presentation, prognosis, and response to therapy in heart disease. Particularly, the phenotypic presentation of patients with non-ischaemic dilated cardiomyopathy (NIDCM) differs between men and women. However, whether the response to mesenchymal stem cell (MSC) therapy is influenced by sex remains unknown. We hypothesize that males and females with NIDCM respond similarly to MSC therapy. METHODS AND RESULTS: Male (n = 24) and female (n = 10) patients from the POSEIDON-DCM trial who received MSCs via transendocardial injections were evaluated over 12 months. Endothelial function was measured at baseline and 3 months post-transendocardial stem cell injection (TESI). At baseline, ejection fraction (EF) was lower (P = 0.004) and end-diastolic volume (EDV; P = 0.0002) and end-systolic volume (ESV; P = 0.0002) were higher in males vs. females. In contrast, baseline demographic characteristics, Minnesota Living with Heart Failure Questionnaire (MLHFQ), and 6-min walk test (6MWT) were similar between groups. EF improved in males by 6.2 units (P = 0.04) and in females by 8.6 units (P = 0.04; males vs. females, P = 0.57). EDV and ESV were unchanged over time. The MLHFQ score, New York Heart Association (NYHA) class, endothelial progenitor cell-colony forming units, and serum tumour necrosis factor alpha improved similarly in both groups. CONCLUSION: Despite major differences in phenotypic presentation of NIDCM in males and females, this study is the first of its kind to demonstrate that MSC therapy improves a variety of parameters in NIDCM irrespective of patient sex. These findings have important clinical and pathophysiologic implications regarding the impact of sex on responses to cell-based therapy for NIDCM.

Clinical Assessment of Intravenous Endothelial Progenitor Cell Transplantation in Dogs.

Endothelial progenitor cells (EPCs) have been applied for cell therapy because of their roles in angiogenesis and neovascularization in ischemic tissue. However, adverse responses caused by EPC therapy have not been fully investigated. In this study, a human peripheral blood sample was collected from a healthy donor and peripheral blood mononuclear cells were separated using Ficoll-Hypaque. There were four experimental groups: 10 ml saline infusion group (injection rate; 3 ml/min), 10 ml saline bolus group (injection rate; 60 ml/min), 10 ml EPCs infusion group (2 x 105 cells/ml, injection rate; 3 ml/min), 10 ml EPCs bolus group (2 × 105 cells/ml, injection rate; 60 ml/min). Clinical assessment included physical examination and laboratory examination for intravenous human EPC transplantation in dogs. The results revealed no remarkable findings in vital signs among the dogs used. In blood analysis, platelet counts in saline infusion groups were significantly higher than in the EPC groups within normal ranges, and no significant differences were observed except K+, Cl- and blood urea nitrogen/urea. In ELISA assay, no significant difference was observed in serum tumor necrosis factor alpha. The serum concentration of vascular endothelial growth factor was significantly higher in EPC groups than in saline groups, and interleukin 10 was significantly up-regulated in the EPC infusion group compared with other groups. In conclusion, we demonstrated that no clinical abnormalities were detected after intravenous transplantation of human EPCs in dogs. The transplanted xenogenic EPCs might be involved in anti-inflammatory and angiogenic functions in dogs.

Japan-United States of America Harmonized Assessment by Randomized Multicentre Study of OrbusNEich's Combo StEnt (Japan-USA HARMONEE) study: primary results of the pivotal registration study of combined endothelial progenitor cell capture and drug-eluting stent in patients with ischaemic coronary disease and non-ST-elevation acute coronary syndrome.

Aims: Harmonized Assessment by Randomized Multicentre Study of OrbusNEich's Combo StEnt (HARMONEE) (NCT02073565) was a randomized pivotal registration trial of the Combo stent, which combined sirolimus and an abluminal bioabsorbable polymer with a novel endoluminal anti-CD34+ antibody coating designed to capture endothelial progenitor cells (EPC) and promote percutaneous coronary intervention (PCI) site healing. Methods and results: Clinically stabilized PCI subjects were randomized 1:1 to receive Combo or everolimus-eluting stents (EES). Between February 2014 and June 2016, 572 subjects with 675 coronary lesions underwent 1-year angiography and fractional flow reserve, with optical coherence tomography (OCT) in the first 140 patients. The primary clinical endpoint was non-inferior 1-year target vessel failure (TVF). The primary mechanistic endpoint of EPC capture activity was superior strut coverage by OCT. Target vessel failure occurred in 7.0% Combo (20/287) vs. 4.2% EES (12/285), a 2.8% [95% confidence interval (95% CI) -1.0%, 6.5%] difference, meeting the non-inferiority hypothesis (P = 0.02). There were no cardiac deaths, with one stent thrombosis observed in the EES group. Quantitative coronary angiography late loss with Combo was equivalent to EES. Optical coherence tomography strut coverage at 1 year was superior with Combo vs. EES [91.3% (95% CI 88.7%, 93.8%) vs. 74.8% (95% CI 70.0%, 79.6%), P < 0.001], with homogeneous tissue in 81.2% vs. 68.8%, respectively. Conclusion: Combo stent demonstrated non-inferior 1-year TVF and late loss in a randomized comparison to EES, with superior strut-based tissue coverage by OCT as a surrogate of EPC capture technology activity.

Assessment of MiRNA Regulation of Endothelial Progenitor Cell Mediated Angiogenesis.

Organ outgrowth, embryonic development, wound healing, and many such processes require the process of angiogenesis, whereby new blood vessels are developed from the preexisting vessels. microRNAs (miRs) are 18-24 nucleotide-containing endogenous RNAs that, via a posttranscriptional mechanism, exert substantial gene regulatory effects. It was discovered by recent advances that, through direct targeting of certain critical secretory factors and transcription factors, miRs exert potent angiogenic control in a cell autonomous and non-cell autonomous manner. This chapter comprehensively summarizes step-by-step protocols for the (1) transfection of miRNA in EPCs (2) advantages and limitations of the principal tubule formation assays in use.

Flt-1 (VEGFR-1) coordinates discrete stages of blood vessel formation.

AIMS: In developing blood vessel networks, the overall level of vessel branching often correlates with angiogenic sprout initiations, but in some pathological situations, increased sprout initiations paradoxically lead to reduced vessel branching and impaired vascular function. We examine the hypothesis that defects in the discrete stages of angiogenesis can uniquely contribute to vessel branching outcomes. METHODS AND RESULTS: Time-lapse movies of mammalian blood vessel development were used to define and quantify the dynamics of angiogenic sprouting. We characterized the formation of new functional conduits by classifying discrete sequential stages-sprout initiation, extension, connection, and stability-that are differentially affected by manipulation of vascular endothelial growth factor-A (VEGF-A) signalling via genetic loss of the receptor flt-1 (vegfr1). In mouse embryonic stem cell-derived vessels genetically lacking flt-1, overall branching is significantly decreased while sprout initiations are significantly increased. Flt-1(-/-) mutant sprouts are less likely to retract, and they form increased numbers of connections with other vessels. However, loss of flt-1 also leads to vessel collapse, which reduces the number of new stable conduits. Computational simulations predict that loss of flt-1 results in ectopic Flk-1 signalling in connecting sprouts post-fusion, causing protrusion of cell processes into avascular gaps and collapse of branches. Thus, defects in stabilization of new vessel connections offset increased sprout initiations and connectivity in flt-1(-/-) vascular networks, with an overall outcome of reduced numbers of new conduits. CONCLUSIONS: These results show that VEGF-A signalling has stage-specific effects on vascular morphogenesis, and that understanding these effects on dynamic stages of angiogenesis and how they integrate to expand a vessel network may suggest new therapeutic strategies.

A quantitative assessment of circulating progenitor cells in competitive athletes and in sedentary subjects.

AIM: Hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs) are known to play a role in the vascular responses and adaptations to exercise. We performed a quantitative assessment of HSCs and EPCs in adolescents in order to investigate whether resting levels of circulating HSCs and EPCs are comparable between elite athletes and sedentary healthy subjects. METHODS: HSCs and EPCs levels were measured in adolescent competitive football players and in age- and sex-matched sedentary controls. A laboratory testing was also performed to determine the white blood cells count and the lipid profile. All athletes were evaluated at the same stage of their training program, after 6 months of training. Controls were not engaged in any kind of routine training program. RESULTS: Twenty male competitive athletes (18.4 ± 0.5 years) and 9 sedentary controls (18.7 ± 0.4 years) participated in the study. As expected, HDL cholesterol was higher in athletes as compared with controls (P<0.05). No significant differences in the other laboratory parameters were observed among groups. Circulating levels of HSCs were significantly lower in athletes in comparison with sedentary controls (P<0.05). Conversely, EPCs and KDR+ cell subpopulations did not substantially differ between athletes and controls. CONCLUSION: Adolescent athletes exhibit lower levels of circulating HSCs but not of EPCs compared to sedentary controls. The process of tissue repair associated with intensive training can contribute to this difference, acting as a stimulus for mobilization and homing of HSCs in the site of injuries.