Physical inactivity promotes the development of cardiovascular diseases. However, few data exist examining the vascular consequences of short-term reductions in daily physical activity. Thus we tested the hypothesis that popliteal and brachial artery flow-mediated dilation (FMD) would be reduced and concentrations of endothelial microparticles (EMPs) would be elevated following reduced daily physical activity. To examine this, popliteal and brachial artery FMD and plasma levels of EMPs suggestive of apoptotic and activated endothelial cells (CD31(+)/CD42b(-) and CD62E(+) EMPs, respectively) were measured at baseline and during days 1, 3, and 5 of reduced daily physical activity in 11 recreationally active men (25 ± 2 yr). Subjects were instructed to reduce daily physical activity by taking <5,000 steps/day and refraining from planned exercise. Popliteal artery FMD decreased with reduced activity (baseline: 4.7 ± 0.98%, reduced activity day 5: 1.72 ± 0.68%, P < 0.05), whereas brachial artery FMD was unchanged. In contrast, baseline (pre-FMD) popliteal artery diameter did not change, whereas brachial artery diameter decreased (baseline: 4.35 ± 0.12, reduced activity day 5: 4.12 ± 0.11 P < 0.05) following 5 days of reduced daily physical activity. CD31(+)/CD42b(-) EMPs were significantly elevated with reduced activity (baseline: 17.6 ± 9.4, reduced activity day 5: 104.1 ± 43.1 per μl plasma, P < 0.05), whereas CD62E(+) EMPs were unaltered. Collectively, our results provide evidence for the early and robust deleterious impact of reduced daily activity on vascular function and highlight the vulnerability of the vasculature to a sedentary lifestyle.

BACKGROUND

Endothelial Microparticles (EMPs) are small vesicles shed from activated or apoptotic endothelial cells and involved in cellular cross-talk. Whether EMP immunophenotypes vary according to stimulus in Diabetes Mellitus (DM) is not known. We studied the cellular adhesion molecule (CAM) profile of circulating EMPs in patients with and without Diabetes Mellitus type 2, who were undergoing elective cardiac catheterization.

RESULTS

EMPs were analyzed by flow cytometry. The absolute median number of EMPs (EMPs/microL) specific for CD31, CD105, and CD106 was significantly increased in the DM population. The ratio of CD62E/CD31 EMP populations reflected an apoptotic process.

CONCLUSIONS

Circulating CD31+, CD105+, and CD106+ EMPs were significantly elevated in patients with DM. EMPs were the only independent predictors of DM in our study cohort. In addition, the EMP immunophenotype reflected an apoptotic process. Circulating EMPs may provide new options for risk assessment.

OBJECTIVE

Spontaneous subarachnoid hemorrhage (SAH) still has a high risk for poor outcome that is frequently attributable to symptomatic cerebral vasospasm (CVS). We hypothesize that cellular microparticles (MP) play a role in the pathogenesis of CVS and may serve as biomarkers for CVS.

METHODS

In 20 consecutive SAH patients, endothelial, leukocyte, platelet, and erythrocyte MP were measured during 15 days after ictus. CVS was detected by transcranial Doppler sonography. Twenty matched volunteers served as healthy controls.

RESULTS

Endothelial, leukocyte, and erythrocyte MP were elevated in SAH patients compared to healthy controls. CD105(+) and CD62e(+) endothelial MP were significantly higher in SAH patients with Doppler sonographic CVS. CD105(+) endothelial MP were especially increased on the days of Doppler sonographic CVS onset. In patients experiencing cerebral infarction attributable to vasospasm, CD41(+) platelet MP were elevated in addition to endothelial MP. CD41(+) platelet MP were significantly higher in patients with any level of disability (modified Rankin Scale score ≥1) compared to those who made a full recovery (modified Rankin Scale score=0) on discharge from hospital.

CONCLUSIONS

Endothelial MP were elevated in patients with SAH. This elevation coincided with the occurrence of Doppler sonographic CVS and therefore could be a novel biomarker for CVS. Platelet MP might be involved in the pathogenesis of cerebral infarction attributable to vasospasm, resulting in neurological morbidity.

OBJECTIVE

Vascular injury plays an important pathophysiological role in vasculitis. Soluble serum thrombomodulin (sTM), a promising marker of endothelial cell injury, is released into the circulating blood following cell damage and was therefore investigated as a parameter of disease activity in patients with Wegener's granulomatosis (WG) and various other vasculitides.

METHODS

One hundred and ninety-seven sera of 102 patients with histologically proven WG of different disease activity and 41 sera of patients with other vasculitides at their active stage were investigated (12 Takayasu arteritis [TA], 7 giant cell arteritis [GCA], 10 polyarteritis nodosa [PAN], 12 Behcet's disease [BD]). The sera were examined for the levels of sTM and sE-selectin (CD62E) by enzyme-linked immunosorbent assay (ELISA) and for the presence of classical anti-neutrophil cytoplasmic antibodies (cANCA) by indirect immunofluorescence (IIF). The disease activity was evaluated according to the clinical symptoms and organ involvement.

RESULTS

A significant increase of sTM levels compared with control values (26 +/- 2 ng/ml) was found in active WG, TA, GCA, PAN, and BD: limited active WG: 63 +/- ng/ml; generalized active WG: 119 +/- 15 ng/ml; limited WG, partial remission: 60 +/- 5 ng/ml; generalized WG, partial remission: 75 +/- 7 ng/ml; active TA: 36 +/-; active GCA: 36 +/- 4 ng/ml, active PAN: 33 +/- 2 ng/ml, active BD: 40 +/- 4 ng/ml. Limited and generalized WG in complete remission did not have elevated levels of sTM. sTM values closely reflected relapses and therapy-induced remissions of WG. Elevated cANCA titers were correlated as well with the disease activity in WG but more weakly than sTM levels. In contrast, sE-selectin values were not significantly correlated with the disease activity and the course of disease.

CONCLUSIONS

sTM is a promising serological marker of disease activity and progression in active limited and generalized WG and other vasculitides reflecting the degree of endothelial cell damage. sTM might prove to be a clinically useful marker for therapeutic considerations.

BACKGROUND

Adhesion of haematopoietic progenitor cells (HPC) to human bone marrow endothelial cells (HBMEC) plays a key role in homing of HPC to bone marrow. Here we describe four new HBMEC cell lines that can be used to study the (specific) adhesion of HPC to HBMEC.

METHODS

HBMEC were immortalised with a retroviral construct containing the human papilloma virus 16 E6/E7 genes. Four cell lines were characterised.

RESULTS

The cell lines showed their endothelial nature by the expression of von Willebrand Factor and VE-cadherin (CD144). Electron microscopic analysis revealed normal endothelial-cell characteristics, including the presence of Weibel-Palade bodies and intercellular junction structures. An extensive phenotypic analysis of the cell-lines was performed, they were found to resemble primary HBMEC. The only difference found was the absence of expression of E-selectin (CD62e) and VCAM-1 (CD106) on resting HBMEC cell lines. Upon stimulation with IL-1beta the expression of E-selectin, VCAM-1 and ICAM-1 (CD54) was upregulated. All resting cell lines bound CD34+ HPC. Adhesion was increased by addition of the phorbol ester PMA. Two cell lines showed increased binding upon IL-1beta prestimulation. Highest adhesion was observed after the combination of IL-1beta prestimulation of the endothelial cells and addition of PMA. Binding of CD34+ HPC to HBMEC was compared with the binding to human umbilical vein endothelial cell lines and to a human dermal microvascular endothelial cell line (HMEC-1). So far, we have only found relatively less binding of HPC to IL-1beta prestimulated HMEC-1 cells, which could be explained by a reduced induction of E-selectin and VCAM-1 upon IL-1beta stimulation of these cells.

CONCLUSIONS

The immortalised HBMEC cell lines have maintained their normal phenotype for the majority of characteristics examined. The expression of E-selectin and VCAM-1, which are not constitutively expressed on the cell lines, can be induced by stimulation of the endothelial cells with IL-1beta. The cell lines have furthermore maintained their capability to bind HPC. They will therefore be useful to investigate the interactions between HPC and HBMEC involved in homing of HPC.

There is a long-standing controversy as to whether a single bone marrow (BM)-derived cell can differentiate along both hematopoietic and stromal lineages. Both primitive hematopoietic and stromal progenitor cells in human BM express the CD34 antigen but lack expression of other surface markers, such as CD38. In this study we examined the CD34+, CD38- fraction of human fetal BM by multiparameter fluorescence-activated cell sorting (FACS) analysis and single-cell sorting. CD34+, C38- cells could be divided into HLA-DR+ and HLA-DR- fractions. After single-cell sorting, 59% of the HLA-DR+ cells formed hematopoietic colonies. In contrast, the CD34+, CD38-, HLA-DR- cells were much more heterogeneous with respect to their light scatter properties, expression of other hematopoietic markers (CD10, CD36, CD43, CD49b, CD49d, CD49e, CD50, CD62E, CD90w, CD105, and CD106), and growth properties. Single CD34+, CD38-, HLA-DR- cells sorted into individual culture wells formed either hematopoietic or stromal colonies. The presence or absence of CD50 (ICAM-3) expression distinguished hematopoietic from stromal progenitors within the CD34+, CD38-, HLA-DR- population. The CD50+ fraction had light scatter characteristics and growth properties of hematopoietic progenitor cells. In contrast, the CD50- fraction lacked hematopoietic progenitor activity but contained clonogenic stromal progenitors at a mean frequency of 5%. We tested the hypothesis that cultures derived from single cells with the CD34+, CD38-, HLA-DR- phenotype could differentiate along both a hematopoietic and stromal lineage. The cultures contained a variety of mesenchymal cell types and mononuclear cells that had the morphologic appearance of histiocytes. Immunophenotyping of cells from these cultures indicated a stromal rather than a hematopoietic origin. In addition, the growth of the histiocytic cells was independent of the presence or the absence of hematopoietic growth factors. Based on sorting more than 30,000 single cells with the CD34+, CD38-, HLA-DR- phenotype into individual culture wells, and an analysis of 864 stromal cultures initiated by single CD34+ BM cells, this study does not support the hypothesis of a single common progenitor for both hematopoietic and stromal lineages within human fetal BM.

BACKGROUND

Most cell types, including blood--and vascular cells, produce microparticles upon activation. Since cellular microparticles are known to be elevated in thromboembolic diseases, we hypothesized a role for microparticles in the pathogenesis of thrombosis in essential thrombocythemia.

METHODS

In plasma samples from 21 patients with essential thrombocythemia and ten healthy subjects, the levels and the cellular origin of microparticles were determined by flowcytometric analysis, while the microparticle-associated procoagulant activity was measured using a thrombin generation assay.

RESULTS

Patients with essential thrombocythemia had significantly higher numbers of circulating annexin V-positive microparticles than controls (median 4500 vs. 2500x10(6) events/L; p=0.039), including significantly higher numbers of microparticles positive for the platelet marker CD61 (p=0.043), the endothelial markers CD62E (p=0.009) and CD144 (p=0.021), and for tissue factor (p=0.036). CD62E was co-expressed with the platelet marker CD41 on microparticles, suggesting a bilineage origin of such microparticles, which were observed only in patients with risk factors for thrombosis. Patients with essential thrombocythemia had higher plasma levels of mature von Willebrand factor (p=0.045) but similar propeptide levels compared to controls. In thrombin generation analyses, microparticle-rich plasma from patients with essential thrombocythemia had a shorter lag time (p=0.001) and higher peak height (p=0.038) than plasma from controls. Peak height correlated significantly with the total number of microparticles (R=0.634, p<0.001).

CONCLUSIONS

Patients with essential thrombocythemia had higher number of circulating microparticles with platelet and endothelial markers, suggesting ongoing platelet and endothelial activation. This was confirmed by an increased level of mature von Willebrand factor, an abnormal mature von Willebrand factor/propeptide ratio, and a hypercoagulable state reflected in thrombin generation. These findings suggest a role for microparticles in thrombosis in essential thrombocythemia.

The mechanisms of the progression of aortic valve stenosis are unknown. The involvement of mononuclear cells and of chronic systemic inflammation has been suggested by analysis of pathological specimens. We hypothesize that shear stress caused by the constricted aortic orifice contributes to systemic proinflammation by activation of circulating blood cells and thereby generation of microparticles. Using flow cytometry we analyzed 22 patients with severe aortic valve stenosis (AVS) and 18 patient controls for the generation of circulating microparticles from platelet- (PMPs: CD31(+)/CD61(+) or CD62P(+)), leukocyte- (LMPs: CD11b(+)) and endothelial cell (EMPs: CD62E(+)) origin. Apart from the constricted valve orifice groups were similar. PMPs were increased in AVS patients and their number correlated with valvular shear stress. Monocytes were activated in AVS patients, an observation that was also reflected by increased numbers of LMPs and by the detection of PMP-monocyte conjugates. Furthermore, EMPs reflecting the activation of endothelial cells but also conferring systemic inflammatory activity were increased in AVS patients and correlated with the number of activated monocytes. In conclusion, we show that AVS is accompanied by increased levels of microparticles and that shear stress can induce the formation of microparticles. Based on our results and histologic findings of other investigators the speculation that shear stress related to aortic valve stenosis induces a vicious circle including the generation of PMPs, the subsequent activation of monocytes and LMPs and finally the activation of endothelial cells contributing to the progress of aortic valve stenosis appears to be justified.

CD18-deficient mice (CD18(-/-) mice) have a severe leukocyte recruitment defect in some organs, and no detectable defect in other models. Mice lacking E-selectin (CD62E(-/-) mice) have either no defect or a mild defect of neutrophil infiltration, depending on the model. CD18(-/-)CD62E(-/-), but not CD18(-/-)CD62P(-/-), mice generated by crossbreeding failed to thrive, reaching a maximum body weight of 10-15 grams. To explore the mechanisms underlying reduced viability, we investigated lethally irradiated CD62E(-/-) mice that were reconstituted with CD18(-/-) bone marrow. These mice, but not single-mutant controls, showed tenfold-increased rolling velocities in a TNF-alpha-induced model of inflammation. Leukocyte adhesion efficiency in CD18(-/-)CD62E(-/-) mice was reduced by 95%, and hematopoiesis was drastically altered, including severe bone marrow and blood neutrophilia and elevated G-CSF and GM-CSF levels. The greatly reduced viability of CD18(-/-)CD62E(-/-) mice appears to result from an inability to mount an adequate inflammatory response. Our data show that cooperation between E-selectin and CD18 integrins is necessary for neutrophil recruitment and that alternative adhesion pathways cannot compensate for the loss of these molecules.

Expression of members of the heat shock protein 60 (hsp60) family in tissues has been reported to coincide with leukocyte infiltration, but it is not known whether these proteins are directly involved in the extravasation of leukocytes. Extravasation of leukocytes requires their adhesion to endothelial cells (EC) via an interaction between adhesion molecules expressed on both cell types. The aim of the present study was to investigate the effect of recombinant mycobacterial hsp65 on the adhesive characteristics of EC for monocytes and granulocytes. Incubation of EC with hsp65 induces a concentration- and time-dependent increase in adhesiveness of these EC for monocytes and granulocytes. The effect was maximal after incubation of EC with hsp65 for 4 to 6 h. In addition, incubation of EC with hsp65 induced the expression of endothelial CD62E (E-selectin), CD106 (vascular cell adhesion molecule-1) and CD54 (intercellular adhesion molecule-1). The increased adhesion of granulocytes to hsp65-stimulated EC was inhibited completely by blocking Ab against CD62E. mAb against endothelial CD62E, CD106, or CD54 or against the monocyte adhesion molecules CD14 or CD49d (very late Ag-4) did not inhibit the increased adhesion of monocytes to hsp65-stimulated EC; however, mAb against the monocyte adhesion molecule CD18 (beta2-integrin) inhibited monocyte adhesion to hsp65-stimulated EC to the same extent as monocyte adhesion to nonstimulated EC. Hsp65 did not exert its effect in an autocrine or paracrine fashion via the endogenous production of IL-1, TNF-alpha, or other factors or via contaminating LPS. Together these results indicate that hsp65 can play an important role in the adhesion of monocytes and granulocytes to EC at sites of inflammation via modulation of the adhesive characteristics of EC and thus may facilitate extravasation of these phagocytes.

The responses of vascular endothelial cells (EC) to tumor necrosis factor-alpha (TNF), interleukin-1alpha (IL-1), and phorbol myristate acetate (PMA) were compared with respect to the kinetics of (i) NF-kappaB activation, (ii) IkappaB-alpha and IkappaB-beta degradation, and (iii) NF-kappaB-dependent cell surface molecule expression. TNF rapidly (</=20 min) and persistently (>20 h) activates NF-kappaB; IL-1 rapidly activates NF-kappaB, but activity declines by 3 h and further by 20 h; PMA slowly and transiently activates NF-kappaB. Untreated EC contain the inhibitory proteins IkappaB-alpha and IkappaB-beta. The onset of NF-kappaB activation correlates with degradation of IkappaB-alpha, but IkappaB-alpha reappears by 4 h without resequestration of NF-kappaB. TNF causes a rapid but partial (50%) reduction in IkappaB-beta, which does not recover by 22 h; IL-1 and PMA cause slower and less sustained reductions in IkappaB-beta. All three agonists induce de novo expression of E-selectin (<em>CD62E</em>) and vascular cell adhesion molecule-1 (CD106) and increase expression of intercellular adhesion molecule-1 (CD54) at 4 h. TNF induces sustained increases in vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 and increases human leukocyte antigen class I molecules at 24 h. We conclude that TNF causes persistent activation of NF-kappaB in human EC and that this may result from sustained reductions in IkappaB-beta levels.

Pulmonary hypertension (PH) is associated with platelet activation, vascular inflammation and endothelial dysfunction leading to often life threatening thrombo-embolic complications. Microparticles (MPs) are cell vesicles with strong coagulatory and inflammatory effects being released during cell activation and apoptosis. As there are currently no established surrogate markers predicting platelet activation and pro-coagulation in PH patients, the aim of the study was to analyze different pro-coagulatory MP populations that might be related to thrombo-embolic complications in PH patients. Circulating MPs from platelet- (PMP, CD31(+)/61(+)), leukocyte- (LMP, CD11b(+)) and endothelial- (EMP, CD62E(+)) origin were measured by flow cytometry in 19 PH patients and were compared to 16 controls. PH patients had increased levels of PMP (PH vs. control 1,016 ± 201 vs. 527 ± 59 counts per min [cpm], P = 0.032), LMP (PH vs. control 31 ± 3 cpm vs. 18 ± 2 cpm, P = 0.001) and EMP (PH vs. control 99 ± 14 cpm vs. 46 ± 6 cpm, P = 0.001). Furthermore, PMP correlated to LMP (PMP vs. LMP: r = 0.75, P < 0.001) and LMP correlated to EMP levels (LMP vs. EMP, r = 0.74, P < 0.001) indicating a functional interaction between the different types of MP. In comparison to non-embolic PH patients, patients with a thrombo-embolic PH suffered from enhanced endothelial cell dysfunction as represented by significantly increased EMP levels (thrombo-embolic PH vs. non-embolic PH 137 ± 27 vs. 72 ± 10, P = 0.02). PH patients have increased levels of platelet-, leukocyte- and endothelial MP indicating an increased vascular pro-coagulation and inflammation which might be related to thrombo-embolic complications as well as PH progression.

Respiratory syncytial virus (RSV) is worldwide the most frequent cause of bronchiolitis and pneumonia in infants requiring hospitalization. In the present study, we supply evidence that human lung microvascular endothelial cells, human pulmonary lung aorta endothelial cells, and HUVEC are target cells for productive RSV infection. All three RSV-infected endothelial cell types showed an enhanced cell surface expression of ICAM-1 (CD54), which increased in a time- and RSV-dose-dependent manner. By using noninfectious RSV particles we verified that replication of RSV is a prerequisite for the increase of ICAM-1 cell surface expression. The up-regulated ICAM-1 expression pattern correlated with an increased cellular ICAM-1 mRNA amount. In contrast to ICAM-1, a de novo expression of VCAM-1 (CD106) was only observed on RSV-infected HUVEC. Neither P-selectin (CD62P) nor E-selectin (CD62E) was up-regulated by RSV on human endothelial cells. Additional experiments performed with neutralizing Abs specific for IL-1alpha, IL-1beta, IL-6, and TNF-alpha, respectively, excluded an autocrine mechanism responsible for the observed ICAM-1 up-regulation. The virus-induced ICAM-1 up-regulation was dependent on protein kinase C and A, PI3K, and p38 MAPK activity. Adhesion experiments using polymorphonuclear neutrophil granulocytes (PMN) verified an increased ICAM-1-dependent adhesion rate of PMN cocultured with RSV-infected endothelial cells. Furthermore, the increased adhesiveness resulted in an enhanced transmigration rate of PMN. Our in vitro data suggest that human lung endothelial cells are target cells for RSV infection and that ICAM-1 up-regulated on RSV-infected endothelial cells might contribute to the enhanced accumulation of PMN into the bronchoalveolar space.

Tumor necrosis factor (TNF) initiates local inflammation by triggering endothelial cells (EC) to express adhesion molecules for leukocytes such as intercellular adhesion molecule-1 (ICAM-1 or CD54). A prior study identified siRNA molecules that reduce ICAM-1 expression in cultured human umbilical vein EC (HUVEC). One of these, ISIS 121736, unexpectedly inhibits TNF-mediated up-regulation of additional molecules on EC, including E-selectin (CD62E), VCAM-1 (CD106) and HLA-A,B,C. 736 siRNA transfection was not toxic for EC nor was there any evidence of an interferon response. 736 Transfection of EC blocked multiple early TNF-related signaling events, including activation of NF-kappaB. IL-1 activation of these same pathways was not inhibited. A unifying explanation is that 736 siRNA specifically reduced expression of mRNA encoding tumor necrosis factor receptor 1 (TNFR1) as well as TNFR1 surface expression. A sequence with high identity to the 736 antisense strand (17 of 19 bases) is present within the 3'UTR of human TNFR1 mRNA. An EGFP construct incorporating the 3'UTR of TNFR1 was silenced by 736 siRNA and this effect was lost by mutagenesis of this complementary sequence. Chemical modification and mismatches within the sense strand of 736 also inhibited silencing activity. In summary, an siRNA molecule selected to target ICAM-1 through its antisense strand exhibited broad anti-TNF activities. We show that this off-target effect is mediated by siRNA knockdown of TNFR1 via its sense strand. This may be the first example in which the off-target effect of an siRNA is actually responsible for the anticipated effect by acting to reduce expression of a protein (TNFR1) that normally regulates expression of the intended target (ICAM-1).

Monocyte migration through the disrupted cerebral endothelial cell (EC) junctions plays an essential role in formation of multiple sclerosis (MS) demyelinating lesions. During pathogenesis of MS, activated ECs release endothelial microparticles (EMP), which possibly facilitate transendothelial migration (TEMIG) of monocytes. To assess functional roles of EMP in MS, specifically, their (i) interaction with monocytes, (ii) effect on monocyte TEMIG in an in vitro model of the brain microvascular endothelial cells (BMVEC), (iii) phenotypic profiles of EMP elicited by MS plasma and (iv) the effects of IFN-beta 1b on release of EMP and on TEMIG of monocytes (mono) and monocytes:EMP complexes (mono:EMP) through the BMVEC. The effect of IFN-beta 1b on the release of EMP and the TEMIG of mono and mono:EMP was assessed by preincubating BMVEC cultures of IFN-beta 1b prior to addition of plasma. Three EMP phenotypes, CD54, CD62E and CD31 were assayed. Plasma specimens from 20 patients with relapsing remitting MS (11 in exacerbation, MS-E, and 9 in remission, ME-R) and 10 healthy controls were studied. Incubation of BMVEC with MS-E plasma yielded elevated levels of EMPCD54, EMP62E and EMPCD31 relative to MS-R and control plasmas. MS-E but not MS-R or control plasma also augmented TEMIG of monocytes, respectively. Mono:EMP complexes further augmented TEMIG relative to mono alone, but only in the presence of MS-E plasma; there was no significant effect with MS-R or control plasmas. The presence of IFN-beta 1b inhibited TEMIG of mono and mono:EMP by 20% and 30%, respectively. MS-E but not MS-R plasma elicited release of activation-derived EMP and enhanced TEMIG of mono and mono:EMP. IFN-beta 1b inhibited TEMIG and release of EMP, suggesting a role of EMP and a novel therapeutic mechanism for IFN-beta 1b in MS.

Mesenchymal stem cells (MSCs) are well known to possess multipotential differentiation and are becoming a good tool for clinical research. However, specific markers for their purification and the mechanism of their osteogenic differentiation remain to be elucidated. In the present study, we compared the expression of CD106, and osteogenic differentiation-related proteins and genes in human bone marrow (BM)-derived MSCs, before and after differentiation by FACS, histochemical staining, immunohistochemical staining, RT-PCR, and real-time PCR. It was found that MSCs were positive for CD13, CD29, CD44, CD73, CD90, CD105, and CD166, but negative for CD14, CD31, CD34, CD62E, CD45, and GlyA. Notably, CD106 was detected before osteogenic induction, but its expression was downregulated 10 fold after 2 weeks of osteogenic differentiation as determined by flow cytometry. The results of RT-PCR and real-time PCR revealed that the expression of CD106 mRNA in MSCs significantly decreased by 7.1-, 4.2-, and 5.1-fold, respectively after osteogenic, chondrogenic, and adipogenic differentiation. In contrast, other MSC-positive markers described above did not change significantly even after differentiation. Compared to levels in control cells, after 2 weeks of osteogenic differentiation, mRNA levels of alkaline phosphatase, bone sialoprotein, osteocalcin, and transcript factors RUNX2 and Osterix showed more than 2-fold, 5-fold, 1.5-fold, 2-fold, and 5-fold increase, respectively. Thus, we speculate that CD106 might be a useful surface marker for BMMSCs. Moreover, alkaline phosphatase, type I collagen, osteonectin, osteopontin, and biglycin were expressed in the early stages of osteogenic differentiation before bone sialoprotein and osteocalcin. The present study should help to provide a novel marker for isolating purified MSCs and characterizing osteogenic differentiation.

An increased number of circulating endothelial cells (CECs) was demonstrated in alpha- and beta-thalassemic patients, beta-thalassemia/hemoglobin E (BE), both splenectomized (BE[S]) and non-splenectomized (BE[NS]), had higher numbers of CECs than alpha-thalassemia, both HbH (alpha-thal l/alpha-thal 2; H) and HbH with hemoglobin Constant Spring (alpha-thal 1/CS; H/CS). CECs were also increased in heterozygous HbE (EA) and homozygous HbE (EE). The highest level of tumor necrosis factor-alpha (TNF-alpha) was found in HbH/CS patients, whereas the highest levels of vascular endothelial growth factor (VEGF) was observed in BE[S] patients. Significant decreases, in protein C and protein S levels were found in both alpha- and beta-thalassemia compared with normal. Good correlations between the numbers of CEC and TNF-alpha, VEGF, protein C, and protein S levels were demonstrated in this study. In addition, markers for endothelial cell activation and injury (intercellular adhesion molecule-1, ICAM-1/CD54; vascular cell adhesion molecule-1, VCAM-1/CD106; and E-selectin, ELAM-1/CD62E) were detected on the surface of isolated CECs using immunofluorescence technique. Appearance of CECs with markers for endothelial cell activation, together with increased levels of TNF-alpha and VEGF and decreased levels of protein C and protein S in the circulation, may account for the propensity of vascular perturbation in thalassemic subjects.

Injection of monosodium urate (MSU) crystals, the etiological cause of gouty arthritis, into murine peritoneal cavities produced an intense recruitment of polymorphonuclear leukocytes (PMN). After 3 mg MSU crystal injection, cell influx was maximal (approximately 10 x 10[6] cells per mouse) at 6 hr postinjection and sustained up to the 24 hr time-point. In mice depleted of mast cells by administration of compound 48/80 72 hr before challenge with MSU crystals a lower PMN influx was measured (58% reduction). The occurrence of endogenous mast cell activation, in the MSU response, was validated by the observation that MSU challenge reduced by more than 90% the number of intact mast cells recovered in the peritoneal washes. Pretreatment of mice with a histamine H1 antagonist (tripolidine; 0.5 mg/kg) or a platelet-activating factor receptor antagonist (WEB2086; 10 mg/kg) significantly reduced by 50 to 60% the number of PMN recovered from the peritoneal cavities. The molecular determinants of this process of leukocyte recruitment were also investigated. Treatment of mice with an anti-CD62P or anti-CD62E monoclonal antibody (mAb; 100 microg i.v.) produced a distinct inhibition of PMN recruitment measured at 6 hr, whereas only a combined administration of both monoclonal antibodies was effective in reducing by 60% the influx of PMN caused by the MSU crystals within 24 hr. In conclusion, these data highlight a role for endogenous mast cells and for endothelial-derived selectins in MSU crystal-induced PMN recruitment into the peritoneal cavity, and may be useful to dissect molecular mechanism(s) which may be operating in gouty arthritis.

BACKGROUND

Work with primary cells is inherently limited by source availability and life span in culture. Flow cytometry offers extensive analytical opportunities but generally requires high cell numbers for an experiment.

METHODS

We have developed assays on a microfluidic system, which allow flow cytometric analysis of apoptosis and protein expression with a minimum number of fluorescently stained primary cells. In this setup, the cells are moved by pressure-driven flow inside a network of microfluidic channels and are analyzed individually by two-channel fluorescence detection. For some assays the staining reactions can be performed on-chip and the analysis is done without further washing steps.

RESULTS

We have successfully applied the assays to evaluate (a) activation of E-selectin (CD62E) expression by interleukin-1beta in human umbilical vein endothelial cells (HUVECs), (b) induction of CD3 by phorbol-12-myristate-13-acetate in freshly prepared human peripheral blood lymphocytes, and (c) staurosporine-induced apoptosis in HUVEC and normal human dermal fibroblasts.

CONCLUSIONS

Results obtained with the microfluidic system are in good correlation with data obtained using a standard flow cytometer, but demonstrate new dimensions in low reagent and cell consumption.

Tumor necrosis factor (TNF) activates transcription of endothelial leukocyte adhesion molecule-1 (CD62E) in endothelial cells (ECs) through the binding to the gene promoter of the p50/p65 heterodimeric form of nuclear factor-kappa B (NF-kappa B) and of the N-terminal phosphorylated form of the ATF2/c-Jun transcription factor, which is phosphorylated by Jun N-terminal kinase (JNK). However, the intracellular signaling pathways that activate endothelial NF-kappa B and JNK in TNF-induced responses are unknown. In this study we have examined the role of a recently described TNF signaling pathway involving sphingomyelin activation to generate ceramide, a potential intracellular mediator. We find that concentrations of TNF that strongly activate NF-kappa B and JNK within 15 minutes do not produce either a measurable decline in sphingomyelin or a measurable generation of ceramide in cultured human umbilical vein ECs at any time examined. Stimulation of ECs with purified sphingomyelinase (SMase) enzyme causes a rapid 60% to 80% decrease in cellular sphingomyelin content and a large increase in ceramide. However, SMase treatment only minimally activates NF-kappa B, achieving levels that are insufficient to initiate gene transcription. Extracellular SMase does not have access to intracellular sphingomyelin, but treatment of ECs with membrane-permeant ceramide analogues still completely fails to activate NF-kappa B and only activates JNK at late times. Neither SMase nor ceramide analogues induce gene transcription or surface expression of endothelial leukocyte adhesion molecules that are readily induced by TNF. Strikingly, low concentrations of membrane-permeant ceramide cause programmed cell death in ECs, a finding not observed at any concentrations of TNF tested. We conclude that ceramide is not an important second messenger for TNF signaling of gene transcription in ECs but may be a second messenger for cell death in response to as-yet-unidentified signals.

A sedentary lifestyle has adverse effects on the cardiovascular system, including impaired endothelial functions. Subjecting healthy men to 7 days of dry immersion (DI) presented a unique opportunity to analyze the specific effects of enhanced inactivity on the endothelium. We investigated endothelial properties before, during, and after 7 days of DI involving eight subjects. Microcirculatory functions were assessed with laser Doppler in the skin of the calf. We studied basal blood flow and endothelium-dependent and -independent vasodilation. We also measured plasma levels of microparticles, a sign of cellular dysfunction, and soluble endothelial factors, reflecting the endothelial state. Basal flow and endothelium-dependent vasodilation were reduced by DI (22 + or - 4 vs. 15 + or - 2 arbitrary units and 29 + or - 6% vs. 12 + or - 6%, respectively, P < 0.05), and this was accompanied by an increase in circulating endothelial microparticles (EMPs), which was significant on day 3 (42 + or - 8 vs. 65 + or - 10 EMPs/microl, P < 0.05), whereas microparticles from other cell origins remained unchanged. Plasma soluble VEGF decreased significantly during DI, whereas VEGF receptor 1 and soluble CD62E were unchanged, indicating that the increase in EMPs was associated with a change in antiapoptotic tone rather than endothelial activation. Our study showed that extreme physical inactivity in humans induced by 7 days of DI causes microvascular impairment with a disturbance of endothelial functions, associated with a selective increase in EMPs. Microcirculatory endothelial dysfunction might contribute to cardiovascular deconditioning as well as to hypodynamia-associated pathologies. In conclusion, the endothelium should be the focus of special care in situations of acute limitation of physical activity.

Traumatic brain injury (TBI) initiates a cascade of acute and chronic injury responses which include disturbances in the cerebrovasculature that may result in the activation of the microvascular endothelial development of a dysfunction endothelium. The present study examines endothelial cell (EC) activation in a percussion model of moderate TBI. The criteria for endothelial activation used in these studies was surface expression of a number of markers collectively termed endothelial activation antigens. Temporal induction of the major histocompatibility (MHC) class II molecules, E-selectin (CD62E), vascular cell adhesion molecule (VACM-1) (CD106) as well as altered expression of constitutively expressed intercellular adhesion molecule-1 (ICAM-1) (CD54), the glucose transporter protein (glut-1), the transferrin receptor (tfR) (CD71), and MHC class I molecules was examined at various times following impact. Induction of E-selectin and increased expression of ICAM-1 was seen by 2 h post-impact (PI) and was sustained through 24 h PI. Decreased expression of immunologically reactive glut-1 and tfR was observed by 2-4 h PI and remained low up to 24 h PI. No induction of VCAM-1, MHC class II molecules or altered constitutive expression or MHC class I molecules was seen. Changes in EC activation were observed predominantly at the site of impact and were diminished temporarily. These results indicate that mild concussive injury to the brain results in activation of the endothelium.

Endothelial cells are part of the normal bone marrow stroma. We have previously shown human umbilical cord blood (UCB) does not produce stroma in standard long-term cultures. Highly enriched (93-98%) UCB CD34+ cells were cultured for 6 weeks with interleukin-2 and conditioned medium from the 5637 carcinoma cell line (n = 4). The resulting 'fibroblast like' cells were shown to be endothelial by expression of von Willebrand factor (VWF), ICAM-1 (CD54), E-selectin (CD62E) and PECAM (CD31). Endothelial monolayers seeded with CD34+ UCB cells supported expansion of colony forming cells and CD34+ cells. We conclude that endothelial cell precursors circulate in UCB, and may be derived from the CD34+ cell fraction.

Endothelial cells express a wide spectrum of surface molecules involved in multiple vascular functions. We quantitatively determined an extensive immunologic phenotype of endothelial cells through a large panel of antibodies directed against i) well-known endothelial molecules CD31, CD34, CD49b, e, f, CD51, CD54, CD55, CD62E and P, CD105, CD106, HLA class I and HLA class II; ii) molecules defined by monoclonal antibodies newly clustered during the 6th workshop of Human Leukocyte Differentiation Antigen (HLDA) CD109, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146 and CD147; iii) molecules defined by unclustered monoclonal antibodies. The expression of these molecules was quantified on human umbilical vein endothelial cells (HUVEC) cultured in resting conditions and after stimulation with IL-1beta (10 U/ml), TNF-alpha (10 ng/ml) and phorbol myristate acetate (60 ng/ml). Some molecules were constitutively expressed, and others were negative, which served to determine the basal phenotype. After cell stimulation, the molecules showed weak or strong expression modulation, leading to the definition of an activated phenotype. Changes in the kinetics and the amplitude of expression served to characterize poorly defined molecules and may be useful to determine their physiologic role. Also, we compared the phenotypes of endothelial cell lines EA.hy 926 and ECV 304 to that of HUVEC to assess their reliability as an endothelial cell model. Each cell line displayed a specific repertoire of molecules expressed at different levels, which could have significant implications for cell line behavior as endothelial cells.