Normal adults have a small number of circulating endothelial cells (CEC) in peripheral blood, and endothelial outgrowth has been observed from cultures of blood. In this study we seek insight into ...the origins of CEC and endothelial outgrowth from cultures of blood. Fluorescence in situ hybridization analysis of blood samples from bone marrow transplant recipients who had received gender-mismatched transplants 5-20 months earlier showed that most CEC in fresh blood had recipient genotype. Endothelial outgrowth from the same blood samples after 9 days in culture (5-fold expansion) was still predominantly of the recipient genotype. In contrast, endothelial outgrowth after approximately 1 month (102-fold expansion) was mostly of donor genotype. Thus, recipient-genotype endothelial cells expanded only approximately 20-fold over this period, whereas donor-genotype endothelial cells expanded approximately 1000-fold. These data suggest that most CEC in fresh blood originate from vessel walls and have limited growth capability. Conversely, the data indicate that outgrowth of endothelial cells from cultures of blood is mostly derived from transplantable marrow-derived cells. Because these cells have more delayed outgrowth but a greater proliferative rate, our data suggest that they are derived from circulating angioblasts.
Background Circulating endothelial cells (CECs) reflect early changes in endothelial health; however, the degree to which CEC number and activation is related to adiposity and cardiovascular risk ...factors in youth is not well described. Methods and Results Youth in this study (N=271; aged 8-20 years) were classified into normal weight (body mass index BMI percentage <85th; n=114), obesity (BMI percentage ≥95th to <120% of the 95th; n=63), and severe obesity (BMI percentage ≥120% of the 95th; n=94) catagories. CEC enumeration was determined using immunohistochemical examination of buffy coat smears and activated CEC (percentage of vascular cell adhesion molecule-1 expression) was assessed using immunofluorescent staining. Cardiovascular risk factors included measures of body composition, blood pressure, glucose, insulin, lipid profile, C-reactive protein, leptin, adiponectin, oxidized low-density lipoprotein cholesterol, carotid artery intima-media thickness, and pulse wave velocity. Linear regression models examined associations between CEC number and activation with BMI and cardiovascular risk factors. CEC number did not differ among BMI classes (
>0.05). Youth with severe obesity had a higher degree of CEC activation compared with normal weight youth (8.3%; 95% CI, 1.1-15.6
=0.024). Higher CEC number was associated with greater body fat percentage (0.02 per percentage; 95% CI, 0.00-0.03
=0.020) and systolic blood pressure percentile (0.01 per percentage; 95% CI, 0.00-0.01
=0.035). Higher degree of CEC activation was associated with greater visceral adipose tissue (5.7% per kg; 95% CI, 0.4-10.9
=0.034) and non-high-density lipoprotein cholesterol (0.11% per mg/dL; 95% CI, 0.01-0.21
=0.039). Conclusions Methods of CEC quantification are associated with adiposity and cardiometabolic risk factors and may potentially reflect accelerated atherosclerosis as early as childhood.
Background We examined feasibility of a unique approach towards gaining insight into heritable risk for early atherosclerosis: surveying gene expression by endothelial cells from living subjects. ...Methods and Results Subjects aged <50 years (mean age, 37; range, 22-49) without obstructive coronary artery disease underwent coronary reactivity testing that identified them as having normal or abnormal coronary endothelial function. Cultures of Blood Outgrowth Endothelial Cells (BOEC) from 6 normal and 13 abnormal subjects passed rigorous quality control and were used for microarray assessment of gene expression. Of 9 genes differentially expressed at false discovery rate <0.1%, we here focus upon abnormal subjects having elevated expression of
(high mobility group box 1) which we unexpectedly found to be linked to low
(laminin gamma 1) expression. This linkage was corroborated by 3 of our past studies and confirmed bio-functionally. Compared with normal BOEC, abnormal BOEC released 13±3-fold more HMGB1 in response to lipopolysaccharide; and they deposited one tenth as much LAMC1 into collagen subendothelial matrix during culture. Clinical follow-up data are provided for 4 normal subjects (followed 13.4±0.1 year) and for 12 abnormal subjects (followed 9.1±4.5 years). Conclusions The known pathogenic effects of high-
and low-
predict that the combination would biologically converge upon the focal adhesion complex, to the detriment of endothelial shear responsiveness. This gene expression pattern may comprise a heritable risk state that promotes early coronary atherosclerosis. If so, the testing could be applied even in childhood, enabling early intervention. This approach offers a way to bridge the information gap between genetics and clinical phenotype.
The vascular pathobiology of sickle cell anemia involves inflammation, coagulation, vascular stasis, reperfusion injury, iron-based oxidative biochemistry, deficient nitric oxide (NO) ...bioavailability, and red cell sickling. These disparate pathobiologies intersect and overlap, so it is probable that multimodality therapy will be necessary for this disease. We have, therefore, tested a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), for efficacy in reducing endothelial activation. We found that pulmonary vascular endothelial VCAM-1 and tissue factor (TF) expression (both are indicators of endothelial activation) are powerfully and significantly inhibited by TSA. This is seen both with pretreatment before the inducing stress of hypoxia/reoxygenation (NY1DD sickle transgenic mouse), and upon longer-term therapy after endothelial activation has already occurred (hBERK1 sickle mouse at ambient air). In addition, TSA prevented vascular stasis in sickle mice, it exhibited activity as an iron chelator, and it induced expression of the antisickling hemoglobin, hemoglobin F. Notably, the TSA analog SAHA (suberoylanilide hydroxaminc acid) that is already approved for human clinical use exhibits the same spectrum of biologic effects as TSA. We suggest that SAHA possibly could provide true, multimodality, salubrious effects for prevention and treatment of the chronic vasculopathy of sickle cell anemia.
Recent research has recognised new populations of non-hematopoietic cells in the blood. One of these, circulating endothelial cells (CECs), often defined by the expression of membrane glycoprotein ...CD146, are rarely found in the blood in health, but raised numbers are present in a wide variety of human conditions, including inflammatory, immune, infectious, neoplastic and cardiovascular disease, and seem likely to be evidence of profound vascular insult. An additional population are endothelial progenitor cells, defined by the co-expression of endothelial and immaturity cell surface molecules and also by the ability to form colonies in vitro. Although increased numbers of CECs correlate with other markers of vascular disease, questions remain regarding the precise definition, cell biology and origin of CECs. For example, they may be damaged, necrotic or apopototic, or alive, and could possess procoagulant and/or proinflammatory properties. However, since these cells seem to be representative of in situ endothelium, their phenotype may provide useful information. Indeed, whatever their phenotype, there is growing evidence that CECs may well be a novel biomarker, the measurement of which will have utility in various clinical settings related to vascular injury. Despite this promise, progress is impeded by the diversity of methodologies used to detect these cells. Accordingly, results are sometimes inconclusive and even conflicting. Nevertheless, increased CECs predict adverse cardiovascular events in acute coronary syndromes, suggesting they may move from being simply a research index to having a role in the clinic. The objective of the present communication is to condense existing data on CECs, briefly compare them with progenitor cells, and summarise possible mechanism(s) by which they may contribute to vascular pathology.
The paucity of organ donors has necessitated redirecting research toward finding alternative means to a heart transplant, such as left ventricular assist devices (LVADs) that will serve not merely as ...bridge-to-transplant but also as destination therapy. To better understand hemorrhagic and thromboembolic complications that currently limit the use of such devices, we studied the endothelial and coagulation system changes in LVAD recipients with time.
We studied these markers of endothelial dysfunction: circulating endothelial cells and expression of E-selectin, vascular cell adhesion molecule, intercellular adhesion molecule, and tissue factor on circulating endothelial cells, thrombin generation (prothrombin fragments 1,2 and thrombin/antithrombin), and fibrinolysis (D-dimer). Our study group consisted of 21 LVAD recipients (on day 0 and on postoperative days 1, 7, 30, 90, and 180) and 7 control patients undergoing non-LVAD cardiac surgery.
Baseline values of intercellular adhesion molecule, E-selectin, tissue factor, thrombin/antithrombin, and D-dimer were significantly higher in LVAD recipients than the normal range. Markers of thrombin generation (thrombin/antithrombin and prothrombin fragments 1,2) and fibrinolysis (D-dimer) peaked postoperatively and declined to baseline levels or below by 3 months. But the expression of inducible endothelial markers (intercellular adhesion molecule, E-selectin, tissue factor) on circulating endothelial cells increased postoperatively, then decreased but remained elevated above preoperative levels for up to 6 months. In our control patients, baseline levels of intercellular adhesion molecule, E-selectin, tissue factor, D-dimer, and thrombin/antithrombin were lower and decreased significantly by day 7, as compared with LVAD recipients (p < 0.05).
Left ventricular assist device recipients experienced significant baseline activation of endothelial and coagulation systems, further accentuated in the early postoperative period. Left ventricular assist device recipients also had prolonged activation of the endothelial and coagulation systems, suggesting activation of the extrinsic (tissue factor) pathway of thrombosis mediated by sustained endothelial dysfunction in these patients. Further studies are needed to determine the clinical influence of such changes in LVAD recipients.
The vessel wall endothelium undoubtedly plays a role in the vascular pathobiology of sickle cell disease. This pilot study tested the feasibility of using an inhibitor of nuclear factor (NF)–κB, a ...transcription factor, to modify the endothelial activation state of patients with this vascular disease. For a total of 7 separate drug exposure tests, 3 subjects with sickle cell disease took sulfasalazine (given orally at 1 g every 8 hours), and the activation state of their circulating endothelial cells (CECs) was assessed using immunofluorescence microscopy. Companion studies were also performed using sulfasalazine in sickle transgenic mice to verify its effect simultaneously on both CECs and vessel wall endothelium. Both CECs and tissue vessel wall endothelium in sickle mice have an activated phenotype. In these mice sulfasalazine significantly reduced CEC expression of vascular cell adhesion molecule (VCAM), intracellular adhesion molecule (ICAM), and E-selectin, and it correspondingly reduced expression of these molecules in some tissue vessels. In humans with sickle cell disease, sulfasalazine significantly reduced CEC expression of VCAM, ICAM, and E-selectin, but it did not reduce expression of tissue factor. Addition of a second transcription factor inhibitor, salsalate, did not change this result. This pilot study suggests that endothelial cell activation state can be modified and down-regulated in vivo by sulfasalazine.
We assessed reproducibility of endothelial microparticles (EMPs) enumeration among youth.
Four microparticle (MP) indices - total MP per microliter platelet free plasma (PFP), total EMPs per ...microliter PFP, percent activated EMPs and percent lactadherin positive (LACT+) of total EMPs - were measured at two visits (baseline and 7 ± 3 days follow-up) to determine reproducibility overall and by obesity status. We examined CD31
or CD144
with CD41-EMP events of size 0.3-1.0 μm. No statistically significant differences were observed between visits for any of the four MP indices. The within-participant and between-participant coefficient of variation was acceptable (range: 1.13-2.37) with good intraclass-correlation coefficient for all indices except total MP per microliter (range: 0.10-1.00).
Total EMPs per microliter PFP, percent-activated EMPs and percent LACT(+) of total EMPs are reproducible among youth.
The vascular wall participates in the pathogenesis of sickle cell disease. To determine whether the endothelium is activated in this disease, we studied the number, origin, and surface phenotype of ...circulating endothelial cells in patients with sickle cell anemia.
We used immunohistochemical examination of buffy-coat smears to enumerate circulating endothelial cells, and we evaluated the surface phenotype by applying preparations of circulating endothelial cells. An immunofluorescence microscopy panel of antibodies was used, including a specific anti-endothelial-cell antibody, P1H12.
Mean (+/-SD) numbers of circulating endothelial cells in normal blood donors, patients with sickle cell trait, and patients with hemolytic anemias not due to hemoglobin S were 2.6+/-1.6, 3.0+/-2.6, and 2.0+/-0.8 per milliliter of whole blood, respectively. Patients with sickle cell anemia who presented with acute painful episodes had 22.8+/-18.2 circulating endothelial cells per milliliter of blood (P<0.001 for the comparison with normal donors), and patients with no such events within one month before or after blood sampling had 13.2+/-11.8 circulating endothelial cells per milliliter of blood (P=0.002 for the comparison with normal donors and P=0.019 for the comparison with patients with acute events). Serial observations of three patients showed a tendency toward higher levels of circulating endothelial cells at the onset of acute painful crises. The average viability of circulating endothelial cells was 66+/-30 percent. In patients with sickle cell anemia, regardless of clinical status, the circulating endothelial cells were predominantly microvascular in origin (CD36-positive), and most of the cells expressed four markers of endothelial-cell activation: intercellular adhesion molecule 1, vascular-cell adhesion molecule 1, E-selectin, and P-selectin.
Our studies suggest that the vascular endothelium is activated in patients with sickle cell anemia, regardless of the patients' clinical status. Adhesion proteins on activated endothelial cells may have a role in the vascular pathology of sickle cell disease.
Objective We characterized the state of the vascular endothelium in pediatric obesity by comparing circulating endothelial cell (CEC) number and activation phenotype in severely obese children to ...that of normal weight, overweight, and obese children. Study design We used immunohistochemical examination of buffy-coat smears to enumerate CEC and immunofluorescence microscopy to quantify activated CEC in 107 children and adolescents. Normal weight (body mass index BMI <85th percentile; n = 40), overweight (BMI 85th-<95th percentile; n = 17), and obese (BMI 95th-<99th percentile; n = 23) participants were recruited from a longitudinal study. Severely obese (BMI ≥99th percentile; n = 27) participants were recruited from a pediatric obesity clinic. Group means (adiposity; systolic blood pressure SBP quartiles) were compared with general linear models, adjusted for sex, age, and race. With Pearson correlations, we characterized relations of CEC with cardiovascular risk factors. Results Activated CEC increased across BMI groups ( P < .002) and SBP quartiles ( P < .05). CEC number and activated CEC were highest in the severely obese group. CEC number was significantly associated with SBP, diastolic blood pressure, and triglycerides level. Activated CEC were significantly associated with SBP and high-density lipoprotein cholesterol levels. Conclusions The vascular endothelium was activated in relation to excess adiposity, particularly in severely obese children, and to elevated SBP in children and adolescents.
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