E-selectin (CD62E, formerly termed ELAM-1) is a cytokine-inducible adhesion molecule which mediates the binding of neutrophils, monocytes, and skin homing T-cells. The murine homologue of E-selectin has been cloned. A monoclonal antibody (21KC10) was used here to study immunohistochemically the expression and regulation of murine E-selectin in vitro and in vivo. As described for the human system, there was no staining of normal endothelium in skin and other tissues. LPS and tumour necrosis factor-alpha (TNF-alpha), but not interleukin-4 (IL-4) or interferon-gamma (IFN-gamma), induced a transient expression of E-selectin, both when injected in vivo and when added to endothelial cell lines in vitro. To analyse temporal expression of E-selectin under pathophysiological conditions in vivo, we chose two murine models of inflammation: allergic (ACD) and irritant contact dermatitis (ICD). Expression of E-selectin was found to be induced on vascular endothelium of post-capillary venules in both ACD and ICD. In ICD, maximal staining of endothelial cells occurred earlier than in ACD. Expression of E-selectin during ICD and ACD was then compared between strains of mice which differ with regard to the intensity of their inflammatory reaction. BALB/c mice, which in contrast to C57BI/6 mice show a denser infiltrate and prolonged influx of granulocyte and monocytes, revealed a more pronounced and more prolonged expression of E-selectin than C57BI/6 mice. This held true for both ACD and ICD, and in each case, peak expression of E-selectin was associated with the highest density of the leukocytic infiltrate. This study thus reveals regulatory mechanisms involved in the expression of murine E-selectin in vivo and in vitro. It also demonstrates a correlation between endothelial expression of E-selectin and the genetically determined intensity of the inflammatory response.

Activation of poly(ADP-ribose) polymerase (PARP) mediates oxidative stress-induced cell injury. We tested the hypothesis that PARP contributes to ischemia-reperfusion (I/R) damage of the liver by triggering the mechanisms of microcirculatory failure. Leukocyte- and platelet-endothelial cell interactions as well as sinusoidal perfusion were analyzed by intravital fluorescence microscopy after lobar hepatic I/R (90 min/30 min) in C57BL/6 x 129/Sv wild-type (PARP+/+) and PARP-deficient (PARP-/-) mice. Hepatic I/R induced leukocyte/platelet-endothelial cell interactions and tissue injury in PARP+/+ mice, as indicated by impaired sinusoidal perfusion and increased alanine aminotransferase (ALT)/aspartate aminotransferase (AST) serum activities. In PARP-/- mice, however, the postischemic increase in the numbers of rolling/adherent leukocytes and platelets was significantly lower. In addition, I/R-induced translocation of CD62P as well as mRNA expression of CD62E, CD54, and CD106 were attenuated. The degree of perfusion failure was reduced and the increase in the ALT/AST activities was lower in PARP-/- mice compared with PARP+/+ mice. We conclude that PARP contributes to hepatic microvascular injury by triggering the expression/translocation of adhesion molecules and modulating leukocyte/platelet-endothelial cell interactions.

BACKGROUND

Inefficient migration of dendritic cells (DC) to regional lymph nodes (LN) upon intracutaneous injection is a major obstacle for effective DC vaccination. Intravenous vaccination is unfavorable, because DC cannot migrate directly from the blood into LN.

METHODS

To enable human monocyte-derived (mo)DC to enter LN directly from the blood, we manipulated them by RNA electroporation to express a human chimeric E/L-selectin (CD62E/CD62L) protein, which binds to peripheral node addressin expressed on high endothelial venules.

RESULTS

Transfection efficiency exceeded 95%, and high E/L-selectin surface expression was detected for >48 h. E/L-selectin RNA-transfected DC displayed an identical mature DC phenotype as mock-transfected DC. Furthermore, E/L-selectin-transfected DC maintained their normal CCR7-mediated migration capacity, and their ability to prime and expand functional cytotoxic T cells recognizing MelanA. Most importantly, E/L-selectin-RNA-transfected DC gained the capability to attach to and roll on sialyl-Lewis(X) in vitro.

CONCLUSIONS

The presented strategy can be readily translated into the clinic, as it involves no stable genetic manipulation or viral transformation, and allows targeting of a large number of LN.

BACKGROUND

The expression of galactose-alpha(1,3)galactose (Gal) on porcine cells represents a major barrier to xenotransplantation. The generation of Gal-/- pigs to overcome this barrier redirected the focus of research to other rejection mechanisms, including cellular immunity. The present in vitro study investigated (1) the adhesive interactions between human leukocyte subsets and primary endothelial cells derived from inbred Gal-/- and Gal+/+ pigs, and (2) the susceptibility of such Gal-/- porcine endothelial cells to human natural killer (NK) cell cytotoxicity.

METHODS

Primary porcine aortic endothelial cells (PAEC) were isolated from Gal-/- (PAEC-Gal-/-) and Gal (PAEC-Gal+/+) pigs. Human peripheral blood mononuclear cells (PBMC), polymorphonuclear neutrophils (PMN), and NK cells were isolated from healthy volunteers and tested in functional adhesion and cytotoxicity assays.

RESULTS

Adhesion of human PBMC, PMN, or purified NK cells on PAEC-Gal-/- cells was not different from that on PAEC-Gal+/+ cells. Comparing the different leukocyte subsets of PBMC, a preferential adhesion of NK and B cells on both PAEC-Gal-/- and PAEC-Gal+/+ was detected. Tumor-necrosis factor-alpha stimulation of PAEC-Gal-/- and PAEC-Gal+/+ induced an increase of CD62E and CD106 expression and increased cellular adhesion, in particular, of PMN. The lack of Gal-/- expression on PAEC-Gal cells did not prevent xenogeneic human NK-cell cytotoxicity mediated by freshly isolated or interleukin-2-activated NK cells.

CONCLUSIONS

Neither human leukocyte adhesion nor xenogeneic NK-cell cytotoxicity against PAEC are impaired by the lack of Gal, indicating that Gal is not a dominant target of cellular rejection.

BACKGROUND

An acute bout of exercise can improve endothelial function and insulin sensitivity when measured on the day following exercise. Our aim was to compare acute high-intensity continuous exercise (HICE) to high-intensity interval exercise (HIIE) on circulating endothelial microparticles (EMPs) and insulin sensitivity in overweight/obese men and women.

METHODS

Inactive males (BMI = 30 ± 3, 25 ± 6 yr, n = 6) and females (BMI = 28 ± 2, 21 ± 3 yr, n = 7) participated in three experimental trials in a randomized counterbalanced crossover design: 1) No exercise control (Control); 2) HICE (20 min cycling @ just above ventilatory threshold); 3) HIIE (10 X 1-min @ ∼ 90% peak aerobic power). Exercise conditions were matched for external work and diet was controlled post-exercise. Fasting blood samples were obtained ∼ 18 hr after each condition. CD62E(+) and CD31(+)/CD42b- EMPs were assessed by flow cytometry and insulin resistance (IR) was estimated by homeostasis model assessment (HOMA-IR).

RESULTS

There was a significant sex X exercise interaction for CD62E(+) EMPs, CD31(+)/CD42b- EMPs, and HOMA-IR (all P < 0.05). In males, both HICE and HIIE reduced EMPs compared to Control (P ≤ 0.05). In females, HICE increased CD62E(+) EMPs (P < 0.05 vs. Control) whereas CD31(+)/CD42b- EMPs were unaltered by either exercise type. There was a significant increase in HOMA-IR in males but a decrease in females following HIIE compared to Control (P<0.05).

CONCLUSIONS

Overweight/obese males and females appear to respond differently to acute bouts of high-intensity exercise. A single session of HICE and HIIE reduced circulating EMPs measured on the morning following exercise in males but in females CD62E(+) EMPs were increased following HICE. Next day HOMA-IR paradoxically increased in males but was reduced in females following HIIE. Future research is needed to investigate mechanisms responsible for potential differential responses between males and females.

BACKGROUND

We investigated vascular endothelial dysfunction by sonographic features of flow-mediated dilation (FMD) and circulating endothelial microparticles (EMPs) in Kawasaki disease (KD).

METHODS

Twenty-eight patients with KD were prospectively grouped according to stage of disease: acute, subacute, and convalescent. In addition, 28 healthy children and 28 febrile children were selected as controls. And cases in the convalescent phase were divided into two subgroups: coronary artery lesion (CAL) and no coronary lesion (NCAL). CD144(+)/CD42b(-), CD62E(+), and CD105(+) EMPs were measured by flow cytometry; FMD was obtained by sonography.

RESULTS

There were significant differences in FMD among the five groups. When compared with healthy controls, there were significantly greater numbers of CD144(+)/CD42b(-), CD62E(+), and CD105(+) EMPs and a higher proportion of CD62E(+) EMPs in KD patients. The proportions and numbers of CD144(+)/CD42b(-), CD62E(+), and CD105(+) EMPs in KD patients were not statistically different than in febrile controls. There were no significant differences in FMD and EMPs between the CAL and NCAL subgroups. There were significantly negative correlations between the values of FMD and EMPs in the three phases of KD.

CONCLUSIONS

The increased levels of EMPs have significant correlation with decreased values of FMD, both of which may reflect endothelial dysfunction in child KD.

OBJECTIVE

To investigate the inflammatory response within intact carotid plaques from carotid eversion endarterectomy (CEE) to determine the relationship between immunohistological plaque morphology and ischaemic cerebrovascular symptoms.

METHODS

Intact CEE plaques from 71 patients with high-grade (>70%) stenosis undergoing CEE (group I, symptomatic, n=42; group II, asymptomatic, n =29) and 12 normal postmortem arteries (control group) were analysed with specific antibodies to inflammatory cells (T-Lymphocytes (CD3, CD4), cytotoxic T-cells (CD8), B-lymphocytes (CD20), natural killer cells (CD57), macrophages (CD68)), endothelial adhesion molecules (ICAM-1 (CD54), P-selectin (CD62P), E-selectin (CD62E), VCAM-1 (CD106) and T-lymphocyte co-stimulatory molecule (CD40)) and procoagulatory modulators (thrombomodulin (CD141), tissue factor (CD142)). Both groups were matched for gender, age, risk factors, degree of carotid artery stenosis. Plaques were measured using a semiquantitative score system in a blinded fashion by two observers. Statistical analysis of the group differences were performed by using the Kruskal-Wallis test and the Multitest Procedure with Permutation-Testing. Significance was taken as a p<0.05.

RESULTS

There were significantly more inflammatory cells, an overexpression of P-selectin and the procoagulatory markers thrombomodulin and tissue factor in symptomatic compared to both asymptomatic plaques and the ones of the control group. In both groups there was no significance for ICAM-1, VCAM-1, macrophages and co-stimulatory molecule CD40. There was also no significance for any factor between the asymptomatic and the control group. However, the differences between the symptomatic and the asymptomatic group were highly significant for all factors.

CONCLUSIONS

These data suggest that structural changes and inflammatory damage within the individual plaque seems to be a critical step in promoting plaque rupture with embolic sequelae.

BACKGROUND

Type two diabetes mellitus (T2DM) remains a leading contributor to cardiovascular mortality worldwide. This study was conducted to investigate the pattern of circulating EMPs in T2DM patients in comparison with MetS subjects.

METHODS

The study retrospectively included 101 patients (54 subjects with T2DM and 47 patients with MetS) and 35 healthy volunteers. All the patients gave written informed consent for participation in the study. Biomarkers were measured at baseline of the study.

RESULTS

There is a significant difference between healthy subjects and patients regarding CD31+/annexin V+ EMPs to CD62E+ EMPs ratio, which reflects impaired phenotype of EMPs. Therefore, CD31+/annexin V+ EMPs to CD62E+ EMPs ratio was found to be higher in the T2DM patients compared to MetS patients. Using multivariate linear regression analyses, independent impact of T2DM (r = 0.40, P = 0.003), OPG (r = 0.37, P = 0.001), hs-CRP (r = 0.347, P = 0.001), and adiponectin (r = 0.33, P = 0.001) on increased CD31+/annexin V+ to CD62E+ ratio of EMPs was determined. Using C-statistics, we found that inflammatory biomarkers (hs-C-reactive protein, osteoprotegerin and adiponectin) added to the base model (T2DM) improved the relative IDI by 12.6 % for increased CD31+/annexin V+ EMPs to CD62E+ EMPs ratio.

CONCLUSIONS

We found that patients with T2DM and MetS may be distinguished by predominantly appearing phenotypes of circulating EMPs associated with pro-inflammatory cytokine overproduction. Elevated CD31+/annexin V+ EMPs to CD62E+ EMPs ratio is an indicator of impaired immune phenotype of EMPs, which allows determining the pattern of EMPs in dysmetabolic disorder patients.

The purpose of the study was to identify characteristics specific to tumor-derived endothelium that may be important in tumor biology, or for the development of targeted therapeutics or imaging agents. Normal C57Bl/6 murine heart or lung endothelium, or C57Bl/6 murine Lewis lung carcinoma tumor-derived endothelium was isolated from excised tissues using specific antibodies. The endothelium was cultured using either native fibronectin, or the oncofetal form of fibronectin. Cell surface adhesion molecule expression was analyzed by flow cytometry, and the cellular distribution of specific molecules was examined using indirect immunofluorescence staining. Oncofetal fibronectin was critical for maintaining the phenotype of tumor-derived endothelium, which demonstrated an elongated morphology in vitro, with few cell-cell contacts. They expressed high levels of CD31, CD102, and vascular endothelial cadherin, and constitutively expressed CD62E, CD54, and CD106, indicating an "activated" phenotype. Moreover, they expressed significantly greater levels of Sca-1 and Flk-1 than normal murine endothelium. Cellular distribution of CD31, beta-catenin, and CD106 in tumor-derived endothelium was not continuous at cell borders, as observed in cultures of murine heart endothelium. In conclusion, Lewis lung carcinoma-derived tumor endothelium exhibits a specific phenotype in vitro, distinct from normal endothelium, and could be used as an in vitro tool for developing targeted agents.

OBJECTIVE

Endothelial microparticles (EMPs) in plasma are elevated in several vascular diseases. Alzheimer's disease (AD) is associated with microcirculatory injury, capillary blocking and disruption of the blood-brain barrier. We wanted to test the hypothesis that EMPs would be increased in AD patients and would correlate with a cognitive decline, and to determine if EMPs are released as a result of activation or apoptosis/necrosis in AD.

METHODS

EMP levels in plasma of AD patients and controls were quantified by flow cytometry. EMP markers for apoptosis/necrosis [platelet/endothelial cell adhesion molecule-1 (PECAM-1)/CD31] and for activation (E-selectin/CD62e) were evaluated. The EMP CD62E/CD31 populations ratio of ≤1.0 was used to differentiate activation from apoptosis.

RESULTS

Significantly higher CD31+/CD42- and CD62e+/CD42- counts were observed in the AD group relative to the controls (p < 0.05). There was no difference between the moderate- to-severe AD group and the mild AD group. Significant correlations were found between circulating EMP counts and Mini-Mental State Examination and AD Assessment cognition (ADAS-cog) score. Multivariate regression analysis demonstrated the persistence of significant correlations between ADAS-cog score and CD31+/CD42- EMPs.

CONCLUSIONS

The (PECAM-1)/CD31 ratio demonstrated that EMPs were generated via apoptosis/necrosis and not by activation. Certain circulating EMP phenotypes may be associated with a cognitive decline of AD patients. EMP analysis shows a promising contribution to understanding vascular pathophysiology in AD.

Levels of circulating endothelial progenitor cells (EPCs) in acute ST-elevation myocardial infarction (STEMI) patients undergoing primary coronary intervention (PCI) were investigated in this study. Flow cytometric analysis of the circulating EPC level (CD31/CD34 [E(1)], CD62E/CD34 [E(2)], and KDR/CD34 [E(3)]) was determined from blood samples of 161 consecutive patients with STEMI undergoing primary PCI. Angiogenesis was evaluated using mononuclear cell-derived EPCs on Matrigel. The EPC number (E(1-3)) was lower in STEMI patients than in normal subjects (n = 25) (P < 0.005). Patients with high EPCs (E(1-3)) (≥1.2%) had a lower left ventricular ejection fraction, elevated white blood cell count and creatinine level, advanced Killip score (≥class 3), more advanced congestive heart failure (CHF) (≥class 3), and increased 30-day mortality than those with a low EPC (E(1-3)) level (<1.2%) (P < 0.0001). Angiogenesis was lower in patients with a high EPC level than those with a low EPC level and normal controls (P < 0.001). Both the advanced Killip score and the CHF were independent predictors of increased EPC levels (P < 0.05). Multivariate analysis identified a high EPC (E(3)) level to be the most important predictor of increased 30-day major adverse clinical outcome (MACO) (P < 0.0001). In conclusion, the circulating EPC level is a major independent predictor of 30-day MACO in patients with STEMI undergoing primary PCI.

Burkholderia pseudomallei induces the formation of multinucleated giant cells in cell monolayers. After infection of human macrophage-like U937 cells with B. pseudomallei, addition of monoclonal antibodies against integrin-associated protein (CD47), E-selectin (CD62E), a fusion regulatory protein (CD98), and E-cadherin (CD324) suppressed multinucleated giant cells in a concentration-dependent manner while monoclonal antibodies against other surface molecules did not inhibit fusion despite binding to the cell surface. Flow cytometric analysis showed increased expression of CD47 and CD98, but not CD62E and CD324, upon B. pseudomallei infection. Our data suggest the involvement of specific cellular factors in the process of B. pseudomallei-induced fusion.

BACKGROUND

15% of reproducing couples suffer from pregnancy loss(PL) and recurs in 2-3%. One of the most frequently hypothesized causes of unexplained PL refers to a defective maternal haemostatic response leading to uteroplacental thrombosis. Hereditary thrombophilia and antiphospholipid antibodies have been extensively described as risk factors for PL in women with unknown aetiology. Recently, a new marker has emerged: the cell-derived procoagulant circulating microparticles(MPs) which have been reported to have a major role in many thrombosis complicated diseases. This study aims to analyze the significance of procoagulant MPs in women suffering from unexplained recurrent pregnancy loss(RPL), and characterize their cellular origin.

RESULTS

115 women with RPL were analyzed for common thrombophilia markers and different cell derived MPs-total annexinV, platelet(CD41a), endothelial(CD146,CD62e), leukocyte(CD45), erythrocyte(CD235a) and tissue factor(CD142)(TF) expressing MPs and were compared with 20 healthy non-pregnant women. Methodology for MP analysis was standardized by participating in the "Vascular Biology Scientific and Standardization Committee workshop".

RESULTS

Total annexinV, TF and endothelial MPs were found significantly increased(p<0.05, 95% confidence interval) in women with RPL. The procoagulant activity of MPs measured by STA-PPL clotting time assay was found in correspondence with annexinV MP levels, wherein the clot time was shortened in samples with increased MP levels. Differences in platelet, leukocyte and erythrocyte derived MPs were not significant. Thirty seven of 115 women were found to carry any of the acquired or hereditary thrombophilia markers. No significant differences were seen in the MP profile of women with and without thrombophilia marker.

CONCLUSIONS

The presence of elevated endothelial, TF and phosphatidylserine expressing MPs at a distance (at least 3 months) from the PL suggests a continued chronic endothelial damage/activation which may get exaggerated at the onset of pregnancy. The data suggests that MPs may contribute to uteroplacental thrombosis and are associated with the pathogenesis of RPL.

OBJECTIVE

Leukocyte recruitment to the site of inflammation is a key event in a variety of cardiovascular pathologies. Infiltrating neutrophils constitute the first line of defense that precedes a second wave of emigrating monocytes reinforcing the inflammatory reaction. The mechanisms initiating this sequential process remained largely obscure.

RESULTS

Using advanced in vivo microscopy and in vitro/ex vivo techniques, we identified individual spatiotemporal expression patterns of selectins and their principal interaction partners on neutrophils, resident/inflammatory monocytes, and endothelial cells. Coordinating the intraluminal trafficking of neutrophils and inflammatory monocytes to common sites of extravasation, selectins assign different sites to these immune cells for their initial interactions with the microvascular endothelium. Whereas constitutively expressed leukocyte L-selectin/CD62L and endothelial P-selectin/CD62P together with CD44 and P-selectin glycoprotein ligand-1/CD162 initiate the emigration of neutrophils, de novo synthesis of endothelial E-selectin/CD62E launches the delayed secondary recruitment of inflammatory monocytes. In this context, P-selectin/CD62P and L-selectin/CD62L together with P-selectin glycoprotein ligand-1/CD162 and CD44 were found to regulate the flux of rolling neutrophils and inflammatory monocytes, whereas E-selectin/CD62E selectively adjusts the rolling velocity of inflammatory monocytes. Moreover, selectins and their interaction partners P-selectin glycoprotein ligand-1/CD162 and CD44 differentially control the intraluminal crawling behavior of neutrophils and inflammatory monocytes collectively enabling the sequential extravasation of these immune cells to inflamed tissue.

CONCLUSIONS

Our findings provide novel insights into the mechanisms initiating the sequential infiltration of the perivascular tissue by neutrophils and monocytes in the acute inflammatory response and might thereby contribute to the development of targeted therapeutic strategies for prevention and treatment of cardiovascular diseases.

OBJECTIVE

Circulating endothelial microparticles act as biological markers of endothelial function that reflect vascular injury. Here, we examined the hypothesis that the quantity of endothelial microparticles in the circulation is increased in patients with ischemic cerebrovascular diseases, and investigated the potential utility of various phenotypes of endothelial microparticles as specific biomarkers of endothelial cell dysfunction. We additionally focused on identifying endothelial microparticles that may be effectively utilized as biomarkers of stroke severity in acute ischemic stroke patients.

METHODS

In total, 129 subjects, including 68 consecutive patients with acute ischemic stroke and 61 age- and sex-matched healthy controls, were included in the study. Levels of circulating endothelial microparticles (CD144+/CD41a-, CD31+CD41a-, CD62E+, Annexin V+CD62E+) and platelet-derived microparticles (CD41a+/CD144-) in platelet-free plasma of patients and controls were measured using flow cytometry.

RESULTS

Levels of circulating endothelial CD144+/CD41a-, CD31+CD41a-, CD62E+, and Annexin V+CD62E+microparticles, but not platelet microparticles, were significantly increased in acute ischemic stroke patients, compared with control subjects (p<0.05). Notably, levels of CD144+/CD41a- microparticles were significantly correlated with stroke severity. A mild degree of correlation was evident between Annexin V+CD62E+microparticles and stroke subtype. No association with stroke was observed for other microparticle phenotypes.

CONCLUSIONS

Circulating endothelial microparticle amounts are increased in acute ischemic stroke patients, compared with healthy subjects. Levels of CD144+/CD41a- microparticle, but not the other phenotypes examined, may be effectively utilized as a biomarker of ischemic severity in the clinic.

BACKGROUND

Microvesicles are small vesicles expressing specific antigens from their cells of origin. Elevated levels of microvesicles have been shown to be associated with coagulation disorders as well as with different types of malignancies. This study aims to evaluate a possible correlation of different microvesicle subpopulations with a positive history of venous thromboembolism (VTE) in patients with soft tissue sarcoma.

METHODS

Annexin V - positive microvesicles, leukocyte (CD45-positive), platelet (CD61-positive), activated platelet (CD62P-, CD63-positive), endothelium-derived (CD62E-positive) and tissue-factor (CD142-positive) microvesicles were identified in the peripheral blood of patients with soft tissue sarcoma (n = 39) and healthy controls (n = 17) using fluorescence-activated cell sorting (FACS).

RESULTS

Both the total amount of Annexin V-positive microvesicles and levels of endothelium-derived (CD62E-positive) microvesicles were shown to decrease significantly after tumor resection (n = 18, p = 0.0395 and p = 0.0109, respectively). Furthermore, the total amount of Annexin V - positive microvesicles as well as leukocyte (CD45-positive) and endothelium-derived (CD62E-positive) microvesicles were significantly higher in patients with grade 3 (G3) soft tissue sarcoma (n = 9) compared to healthy controls (n = 17) (p = 0.0304, p = 0.0254 and p = 0.0357, respectively). Moreover, patients with G3 soft tissue sarcoma (n = 9) presented higher levels of Annexin V-positive and endothelium-derived (CD62E-positive) microvesicles compared to patients with grade 2 (G2) soft tissue sarcoma (n = 8) (p = 0.0483 and p = 0.0045). Patients with grade 1 (G1) soft tissue sarcoma (n = 3) presented with significantly lower levels of platelet (CD61-positive) microvesicles than patients with G3 soft tissue sarcoma (n = 9) (p = 0.0150). In patients with a positive history of VTE (n = 11), significantly higher levels of activated platelet (CD62P- and CD63-positive) microvesicles (p = 0.0078 and p = 0.0450, respectively) were found compared to patients without a history of VTE (n = 28).

CONCLUSIONS

We found significantly higher levels of Annexin V-positive and endothelium-derived (CD62E-positive) microvesicles to be circulating in the peripheral blood of patients with G3 soft tissue sarcoma compared to patients with G2 soft tissue sarcoma. Furthermore, we showed that high counts of activated platelet-derived microvesicles correlate with the occurrence of VTE. Thus, the detection of these microvesicles might be an interesting new tool for early diagnosis of soft tissue sarcoma patients with increased risk for VTE, possibly facilitating VTE prevention by earlier use of thromboprophylaxis.

Reducing excessive oxidative stress, through chronic exercise or antioxidants, can decrease the negative effects induced by excessive amounts of oxidative stress. Transient increases in oxidative stress produced during acute exercise facilitate beneficial vascular training adaptations, but the effects of non-specific antioxidants on exercise training-induced vascular adaptations remain elusive. Circulating angiogenic cells (CACs) are an exercise-inducible subset of white blood cells that maintain vascular integrity. We investigated whether mitochondria-specific antioxidant (MitoQ) supplementation would affect the response to 3 weeks of endurance exercise training in CACs, muscle mitochondrial capacity and maximal oxygen uptake in young healthy men. We show that endurance exercise training increases multiple CAC types, an adaptation that is not altered by MitoQ supplementation. Additionally, MitoQ does not affect skeletal muscle or whole-body aerobic adaptations to exercise training. These results indicate that MitoQ supplementation neither enhances nor attenuates endurance training adaptations in young healthy men.

Antioxidants have been shown to improve endothelial function and cardiovascular outcomes. However, the effects of antioxidants on exercise training-induced vascular adaptations remain elusive. General acting antioxidants combined with exercise have not impacted circulating angiogenic cells (CACs). We investigated whether mitochondria-specific antioxidant (MitoQ) supplementation would affect the response to 3 weeks of endurance exercise training on CD3+ , CD3+ /CD31+ , CD14+ /CD31+ , CD31+ , CD34+ /VEGFR2+ and CD62E+ peripheral blood mononuclear cells (PBMCs), muscle mitochondrial capacity, and maximal oxygen uptake (VO2 max ) in healthy men aged 22.1 ± 0.7 years, with a body mass index of 26.9 ± 0.9 kg m-2 , and 24.8 ± 1.3% body fat. Analysis of main effects revealed that training induced 33, 105 and 285% increases in CD14+ /CD31+ , CD62E+ and CD34+ /VEGFR2+ CACs, respectively, and reduced CD3+ /CD31- PBMCs by 14%. There was no effect of MitoQ on CAC levels. Also independent of MitoQ supplementation, exercise training significantly increased quadriceps muscle mitochondrial capacity by 24% and VO2 max by roughly 7%. In conclusion, endurance exercise training induced increases in multiple CAC types, and this adaptation is not modified by MitoQ supplementation. Furthermore, we demonstrate that a mitochondrial-targeted antioxidant does not influence skeletal muscle or whole-body aerobic adaptations to exercise training.

Nonmetastatic gene 23-H1 (NME1, also known as nm23-H1) is a wide-spectrum tumor metastasis suppressor gene that plays an important role in suppressing the proliferation, adhesion and invasion of endometrial stromal cells (ESCs). The present study is undertaken to explore the mechanism by which NME1 in ESCs from endometriosis modulates the angiogenesis and herein participates in the pathogenesis of endometriosis. The expression of NME1 in the primary ESCs from normal endometrium without endometriosis was higher than that from eutopic endometrium and ectopic lesion with endometriosis. Silencing NME1 stimulated the secretion of angiogenic factors interleukin-8 (IL-8) and vascular-endothelial growth factor (VEGF) of the eutopic ESCs from women with endometriosis, and these effects could be abrogated by MAPK/ERK1/2 or AKT inhibitor. In addition, the supernatant of NME1-silenced ESCs increased the expression of angiogenesis-relative molecules CD62E and CD105, and promoted angiogenesis of human umbilical vein endothelial cells (HUVECs). Anti-human IL-8 or VEGF neutralizing antibody reversed the effect on angiogenesis of HUVECs induced by NME1-silenced ESCs. Our current results suggest that the abnormal lower expression of NME1 in ESCs secrete more IL-8 and VEGF through activation of MAPK/ERK1/2 and AKT signal pathways, up-regulate the level of CD62E and CD105, and finally lead to numerous angiogenesis of vascular endothelial cells in the endometriotic milieu, which is beneficial to the origin and development of endometriosis.

E-selectin (CD62E) is a cytokine-inducible endothelial cell adhesion molecule that tethers polymorphonuclear leukocytes (PMNs) and supports PMN rolling under conditions of flow. We examined whether interaction of PMNs with E-selectin also leads to activation of CD11b/CD18 (Mac-1, alphaMbeta2), an event that can promote firm adhesion. PMNs were added to monolayers of IL-1beta-activated HUVECs and Chinese hamster ovary (CHO) cells transfected with E-selectin cDNA. PMN activation was assessed by 1) increased CD11b/CD18 surface expression, 2) appearance of activation epitope on CD11b/CD18 (CD11b*) detected by mAb CBRM1/5, and 3) decreased L-selectin (CD62L) expression, as determined by flow cytometry. Both adherent and nonadherent supernatant PMNs became activated on IL-1beta-pretreated HUVECs. This activation was not affected by CD62E-blocking mAb P6E2. The activation state of PMNs adhered to CHO cells transfected with E-selectin cDNA was not increased over background and was similar to that of PMNs exposed to parent CHO cells. The findings were confirmed using confocal microscopy, which allowed staining of the cells for CD11b* in situ. In concert, the results suggest that PMN binding to E-selectin does not elicit inter-receptor signaling that could result in strengthening of PMN adhesion to endothelium.

BACKGROUND

Prostate growth seems to be influenced by paracrine factors like IL-6 originating from the microvascular endothelium. Therefore, our efforts were focused on the primary culture and behavior of microvascular endothelial cells (HPEC) derived from tissue of human benign prostatic hyperplasia (BPH). Until now, the isolation and culture of HPEC from BPH have not been reported.

METHODS

BPH tissue was cut into small cubes and gently squeezed after incubation with dispase. HPEC were cultured from the resulting cell suspension after a stepwise selection by use of superparamagnetic beads coated with antibodies against endothelial specific antigens. HPEC were characterized by flow cytometry and immunohistochemistry. gamma-Glutamyl transpeptidase activity (specific for microvascular endothelium) was measured after dissolution of the HPEC with Triton X-100. After the incubation of HPEC either with ATP, VEGF, or TNF-alpha, the release of IL-6 was measured by enzyme linked immunosorbent assay (ELISA).

RESULTS

HPEC showed a typical endothelial morphology. They were positive for von Willebrand factor, CD31, CD62E (after stimulation with TNF-alpha), alpha-actin and were negative for fibroblastic antigens and PSA. Proliferation was stimulated by vascular endothelial growth factor (VEGF). gamma-Glutamyl transpeptidase activity in HPEC was 6.3 microIU/microg protein, whereas in human umbilical vein endothelial cells (HUVEC) no gamma-glutamyl transpeptidase activity was detectable. The IL-6 secretion of HPEC was stimulated by VEGF and TNF-alpha, but not by ATP and bradykinin.

CONCLUSIONS

For the first time, the primary culture of microvascular endothelial cells from BPH tissue was successfully performed. Our results suggest that HPEC may be actively involved in prostate growth, due to the secretion of regulatory factors such as IL-6.

BACKGROUND

Loss of the pulmonary microvasculature in the pathogenesis of emphysema has been put forward as a credible alternative to the classical inflammatory cell driven proteolysis hypothesis. Mechanistic studies in this area have to date employed animal models, immortalised cell lines, primary endothelial cells isolated from large pulmonary arteries and non-pulmonary tissues and normal human pulmonary microvascular endothelial cells. Although these studies have increased our understanding of endothelial cell function, their relevance to mechanisms in emphysema is questionable. Here we report a successful technique to isolate and characterise primary cultures of pulmonary microvascular endothelial cells from individuals with severe emphysema.

METHODS

A lobe of emphysematous lung tissue removed at the time of lung transplantation surgery was obtained from 14 patients with severe end-stage disease. The pleura, large airways and large blood vessels were excised and contaminating macrophages and neutrophils flushed from the peripheral lung tissue before digestion with collagenase. Endothelial cells were purified from the cell mixture via selection with CD31 and UEA-1 magnetic beads and characterised by confocal microscopy and flow cytometry.

RESULTS

Successful isolation was achieved from 10 (71%) of 14 emphysematous lungs. Endothelial cells exhibited a classical cobblestone morphology with high expression of endothelial cell markers (CD31) and low expression of mesenchymal markers (CD90, αSMA and fibronectin). E-selectin (CD62E) was inducible in a proportion of the endothelial cells following stimulation with TNFα, confirming that these cells were of microvascular origin.

CONCLUSIONS

Emphysematous lungs removed at the time of transplantation can yield large numbers of pulmonary microvasculature endothelial cells of high purity. These cells provide a valuable research tool to investigate cellular mechanisms in the pulmonary microvasculature relevant to the pathogenesis of emphysema.

BACKGROUND

Xenotransplantation of pig organs may provide an approach to alleviate the severe shortage of human organs. Natural antibodies against Galalpha(1,3)-Gal (alphaGal) epitopes cause hyperacute rejection of pig organs in primates. However, evidence for the role of alphaGal in the natural killer (NK) cell-mediated xenoresponse has been contradictory.

METHODS

We investigated the recognition of alphaGal by human NK cells using endo-beta-galactosidase C, an enzyme that cleaves alphaGal, and endothelial cells (EC) from alpha1,3-galactosyltransferase null pigs that do not synthesize alphaGal. Endo-beta-galactosidase C treatment variably reduced the susceptibility of porcine EC to lysis by fresh human NK cells.

RESULTS

Removal of alphaGal from porcine EC using endo-beta-galactosidase C, produced variable results, i.e. cytotoxicity was decreased in half of the human NK cell donors tested. The two EC strains from alphaGal-/- pigs were marginally, and not significantly, less susceptible to lysis by naïve human NK cells compared with alphaGal-expressing cells obtained from animals from the same herd, but these differences were not statistically significant (P>> 0.10). Treatment of porcine EC with recombinant human tumor necrosis factor (TNF)-alpha, which is known to activate porcine EC, enhanced the susceptibility of all target cells to lysis by fresh human NK cells. Surface expression of MHC or adhesion molecules on alphaGal-/- cells, compared with wild type cells, showed no consistent difference in either MHC or adhesion molecules CD106 (VCAM-1), CD31 (PECAM) or CD62E (E-selectin), either with or without TNF-alpha stimulation, that could explain the differential susceptibility to lysis. Strikingly, all alphaGal-/- and wild type EC exhibited similar susceptibility to human NK cells that had been cultured for 5 days with or without interleukin-2.

CONCLUSIONS

These findings demonstrate that human NK cells can kill porcine targets in the absence of alphaGal, and donor variability plays a major role in whether alphaGal has a role in determining susceptibility of porcine EC to lysis. Moreover, susceptibility to lysis of alphaGal null EC is enhanced to the level of wild type EC by activation of either effector or target cells. Elimination of alphaGal alone from source pigs will be insufficient to circumvent the NK cell mediated destruction of porcine EC.

BACKGROUND

The efficacy of highly active antiretroviral therapy (HAART) determined by simultaneous monitoring over 100 cell-surface antigens overtime has not been attempted. We used an antibody microarray to analyze changes in the expression of 135 different cell-surface antigens overtime on PBMC from HIV+ patients on HAART. Two groups were chosen, one (n = 6) achieved sustainable response by maintaining below detectable plasma viremia and the other (n = 6) responded intermittently. Blood samples were collected over an average of 3 years and 5-8 time points were selected for microarray assay and statistical analysis.

RESULTS

Significant trends over time were observed for the expression of 7 cell surface antigens (CD2, CD3epsilon, CD5, CD95, CD36, CD27 and CD28) for combined patient groups. Between groups, expression levels of 10 cell surface antigens (CD11a, CD29, CD38, CD45RO, CD52, CD56, CD57, CD62E, CD64 and CD33) were found to be differential. Expression levels of CD9, CD11a, CD27, CD28 and CD52, CD44, CD49d, CD49e, CD11c strongly correlated with CD4+ and CD8+ T cell counts, respectively.

CONCLUSIONS

Our findings not only detected markers that may have potential prognostic/diagnostic values in evaluating HAART efficacy, but also showed how density of cell surface antigens could be efficiently exploited in an array-like manner in relation to HAART and HIV-infection. The antigens identified in this study should be further investigated by other methods such as flow cytometry for confirmation as biological analysis of these antigens may help further clarify their role during HAART and HIV infection.

BACKGROUND

Impaired maternal immune tolerance resulting in systemic inflammation plays a pivotal role in the pathogenesis of preeclampsia. Phenotypical changes of monocytes and neutrophil granulocytes have already been studied in preeclampsia, and some studies also included T lymphocyte activation markers; however, the results are controversial and a comprehensive analysis of activation markers is lacking. The characteristics of cellular adhesion molecules in preeclampsia are yet to be described.

METHODS

Peripheral blood samples of 18 preeclamptic patients and 20 healthy pregnant women in the third trimester were evaluated using flow cytometry to characterize the cell surface expression of T lymphocyte activation markers and selectins.

RESULTS

We found an elevated ratio of HLA-DR and CD122-, CD62E-, and CD62L-expressing cells among the CD4+ T lymphocytes in PE in comparison to healthy pregnancy. No alterations were found in the prevalence of CD69-, CD25-, and CD62P-expressing lymphocytes and CD11c-expressing monocytes.

CONCLUSIONS

Our findings support the role of activated T lymphocytes and specific cell adhesion molecules in the pathogenesis of preeclampsia.