The leukocyte adhesion deficiency syndrome type II (LAD-II) is caused by a general defect in fucose metabolism, which leads to the absence of fucosylated sugar determinants such as the selectin ligand SLe(x). In view of the important role of selectins in lymphocyte migration and homing, we have explored the in vivo immune responsiveness and lymphocyte recruitment to the skin, in response to the neo-antigen keyhole limpet hemocyanin (KLH) in a LAD-II patient. We observed a normal priming of KLH-specific T cells as well as a strong in vivo anti-KLH antibody response, both indicative of a normal T-B cell function and collaboration. Skin biopsies from the patient's skin taken prior to antigenic challenge showed the presence of normal numbers and subsets of T cells. Upon KLH injection, a large number of T cells were found to be recruited to the site of challenge, which was paralleled by up-regulation of the endothelial adhesion molecules ICAM-1 (CD54), VCAM-1 (CD106) and E-selectin (CD62E). The recruited T cell showed a normal subset distribution but lacked cutaneous lymphocyte antigen (CLA), the cutaneous homing receptor. However, the clinical symptoms of delayed-type hypersensitivity in the patient (redness and swelling) were severely depressed compared to that in the controls. In conclusion, the LAD-II patient showed a normal T cell priming and T cell-dependent antibody response, a normal baseline skin homing, and a significant T cell recruitment to the site of KLH challenge, indicating that fucosylated sugar determinants such as SLe(x) and CLA are not strictly required for adequate immune responsiveness and (skin) homing.

OBJECTIVE

Circulating levels of endothelial cell adhesion molecules are elevated in women with preeclampsia. The aim of the present study was to determine levels of these molecules in the fetal circulation of normal pregnancies and pregnancies complicated by preeclampsia.

METHODS

Fetal plasma samples from the umbilical vein and peripheral maternal plasma and serum sample were collected at delivery from women with preeclampsia and women with normal pregnancy. Women with non-proteinuric pregnancy-induced hypertension (PIH) were excluded from the study. A sandwich ELISA technique was employed to quantitate concentrations of soluble ICAM-1 (CD54), VCAM-1 (CD106), and E-selectin (CD62E).

RESULTS

The normal values of soluble endothelial cell adhesion molecules in the fetal circulation were determined as 162+/-45 ng/ml for ICAM-1, 1612+/-582 ng/ml for VCAM-1, and 154+/-58 ng/ml for E-selectin. They were found to markedly differ from the corresponding normal values in the maternal circulation (sICAM-1: 247+/-65 ng/ml; sVCAM-1: 715+/-170 ng/ml; sE-selectin: 34+/-14 ng/ml). The concentrations of sICAM-1, sVCAM-1, and sE-selectin were significantly elevated in women with preeclampsia compared to healthy control pregnant women. In contrast, there was no difference in the circulating fetal concentrations of these molecules between normal pregnancies and pregnancies complicated by preeclampsia.

CONCLUSIONS

Normal values of sICAM-1, sVCAM-1, and sE-selectin in fetal circulation are markedly different from the values obtained for healthy adults. Plasma concentrations of these molecules are elevated in women with preeclampsia but not in the fetal circulation of preeclamptic pregnancies suggesting that based on the analysis of soluble adhesion molecules the fetal circulation may not be affected by the factor(s) that lead to disturbed endothelial cell function in women with preeclampsia.

BACKGROUND

Acute vascular rejection in pig-to-primate xenotransplantation involves recognition and damage of porcine (po) endothelial cells (EC) by human (hu) leukocytes, probably including natural killer (NK) cells. To study such interactions we analyzed rolling and static adhesion of hu NK cells to po EC.

METHODS

The effects of blocking hu and po adhesion molecules on the adhesion hu NK cells to po EC monolayers was analyzed under shear stress (10 min, 37 degrees C, 0.7 dynes/cm2) or under static conditions (10 min, 37 degrees C). All used cell populations were phenotypically characterized by flow cytometry.

RESULTS

Blocking of CD106 on po EC or its ligand CD49d on hu NK cells decreased rolling adhesion of both fresh and activated hu NK cells by more than 75%. Masking of CD62L on fresh but not activated hu NK resulted in a 44% decrease in rolling adhesion, in line with the diminished cell surface expression of CD62L upon activation. Antibodies to CD31, CD54, CD62E, and CD62P on EC or CD11a, CD18, and CD162 on NK cells had only minor effects on rolling adhesion. The adhesion of the FcgammaRIII- hu NK cell line NK92 to po EC was inhibited by 95% after masking po CD106 whereas antibodies to po CD31, CD54, CD62E, or CD62P had no effect, thereby excluding effects of Fc-receptor-dependent binding of hu NK cells to po EC. Static adhesion of activated NK cells was reduced by approximately 60% by blocking either CD49d or CD106, by 47% by blocking CD11a, and by 82% upon simultaneous blocking of CD11a and CD49d.

CONCLUSIONS

Interactions between hu CD49d and po CD106 are crucial for both rolling and firm adhesion of hu NK cells to po EC and thus represent attractive targets for specific therapeutic interventions to prevent NK cell-mediated responses against po xenografts.

OBJECTIVE

Antimicrobial agents are important risk factors for infusion phlebitis, but the risk varies between different antibiotics. Erythromycin and dicloxacillin are known to induce phlebitis frequently, as well as to exert toxic effects on cultured endothelial cells. The pathogenesis of infusion phlebitis is unclear, but chemical toxicity is thought to lead to inflammation and subsequent thrombosis. In the present study, endothelial cells were exposed to antibiotics at the range of concentrations used for intravenous administration, followed by analysis of pro-inflammatory and pro-coagulant surface molecules.

METHODS

Primary human umbilical vein endothelial cells (HUVEC) and the endothelial hybrid cell line EaHy926 were exposed to dicloxacillin, erythromycin, benzylpenicillin and cefuroxime (all at 6250 mg/L) for 60 min, followed by washing. After 5 or 24 h additional incubation, cells were analysed for E-selectin (CD62E), tissue factor (TF) or intercellular adhesion molecule 1 (ICAM-1, CD54) density by flow cytometry.

RESULTS

Despite constitutive expression of ICAM-1 (34%) in HUVEC, 6250 mg/L of dicloxacillin or erythromycin significantly increased the number of cells with ICAM-1 expression by 37% and 30%, respectively. In contrast, cefuroxime and benzylpenicillin did not up-regulate ICAM-1 above background levels. A similar pattern was seen with the endothelial cell line EaHy926. The E-selectin and TF density were not affected by the antibiotics examined.

CONCLUSIONS

The results of this study support the theory that endothelial cells that are affected by high concentrations of antibiotics may initiate an inflammatory response through expression of ICAM-1. This is a novel finding in the pathogenesis of infusion phlebitis.

BACKGROUND

Recombinant human endostatin (rh-endostatin, Endostar) has been proved to be an inhibitor of angiogenesis. Docetaxel has been also considered as a common chemotherapeutic agent with inhibition of angiogenesis of malignancies. However, their function has been seldom compared and a best synergism protocol is not determined. This study aimed to compare the effects of two drugs, investigate their combined impact on human umbilical vein endothelial cells (HUVECs), a molecular basis and find ideal protocols to inhibit endothelial cell proliferation.

METHODS

HUVECs on confluent growth or activated by vascular endothelial growth factor (VEGF) were treated by rh-endostatin or/and docetaxel at respective gradient concentration in following operations as cell proliferation determined by MTT assay, cell cycle distribution, apoptosis and markers of CD146, CD62E and CD105 detected by flow cytometery, the structure of the channel formed by HUVECs measured by tube formation count.

RESULTS

Rh-endostatin exhibited time dependent inhibition of proliferation while docetaxel showed both time and dose dependent inhibition. HUVECs accumulated in G(0)-G(1) with decreased numbers of cells in G(2) after a single treatment of rh-endostatin or that followed by docetaxel treatment. Cells accumulated in G(2) after both a single docetaxel and simultaneous administration. Both the number of cells in G(0)-G(1) and apoptotic cells were increased by docetaxel followed by rh-endostatin treatment. The number of non-apoptotic cells at G(0)-G(1) was increased by first administering rh-endostatin then docetaxel. Sequential treatment of docetaxel followed by rh-endostatin resulted in the greatest increase in apoptosis (34.7%) and the second highest apoptosis was seen with simultaneous administration (18.2%). Expression of CD146 and CD105 on confluent HUVECs was reduced at certain doses of rh-endostatin and/or docetaxel. However, rh-endostatin reduced CD105 without any apparent impact on either CD146 or CD62E expression, whereas these markers were down-regulated by docetaxel after pre-activation by VEGF. Rh-endostatin treatment maintained tube-like structures for a limited time. In contrast, docetaxel swiftly reduced tube formation. Simultaneous treatment, or docetaxel followed by rh-endostatin, exhibited a stronger inhibition on tube formation than either agent alone.

CONCLUSIONS

Both rh-endostatin and docetaxel can inhibit HUVEC proliferation while the high apoptotic rate after combined administration was probably owing to different sequent administration by docetaxel followed by rh-endostatin or simultaneous treatment. Both proliferation and adhesion molecules on HUVECs of confluent growth are down-regulated by the two drugs. The rh-endostatin decreased proliferation markers, but only slightly modified adhesion molecules, while both markers were down-regulated by docetaxel on HUVECs activated by VEGF. Rh-endostatin could maintain adhesion of HUVECs at first then induce cells apoptosis to damage tube formation. We hypothesize that it could lead to vascular normalization in short time. In contrast, docetaxel can suppress HUVEC proliferation, adhesion, and reduced tube formation swiftly due to its cytotoxicity. Combined treatments can induce a synergistic inhibition of tube formation.

Twenty-two astronauts who flew aboard 10 different US Space Shuttle flights were studied 10 days before launch, on landing day, and 2-4 days post-landing. After landing, plasma levels of norepinephrine (p<0.01) were elevated. Lymphocyte beta(2)-adrenergic receptors were desensitized 2-4 days post-landing (p<0.02). The density of CD62L on lymphocytes was unchanged but the densities of CD11a (p<0.01) and CD54 (p<0.001) were down-regulated. CD11a density was also down-regulated on monocytes (p<0.01). Neutrophils showed an up-regulation of CD11a (p<0.01) and a down-regulation of CD54 (p<0.01). CD11a density on neutrophils remained up-regulated (p<0.01) and CD54 density remained down-regulated (p<0.01) at 2-4 days post-landing. Circulating levels of soluble ICAM-1 (CD54) and soluble E-selectin (CD62E) were decreased after landing (p's<0.05). The data suggest that spaceflight leads to an environment that would support reduced leukocyte-endothelial adhesion. Sympathetic activation may contribute to this phenomenon.

OBJECTIVE

Sialyl Lewis(x) (sLe(x)) is one ligand for E selectin (CD62E), a glycoprotein that is expressed on activated endothelial cells. Adhesion mediated by endothelial E selectin and sLe(x) expressed on human tumor cells could be relevant for the development of metastases. The aim of this study was to investigate whether or not a correlation exists between pre-operative levels of ganglioside serum sLe(x) and disease-free interval and survival in colorectal cancer patients.

METHODS

Thirty Duke's B and 52 Duke's C patients undergoing resection for colorectal cancer were studied. The median follow-up time was 34.8 months. A pool of 57 sera from normal subjects was used as an Internal Normal Standard (INS). sLe(x) analyses were performed by a thin layer chromatography (TLC) immunostaining technique. Results were expressed as the ratio (R) between bands of INS and bands from each neoplastic serum.

RESULTS

The median R value was 0.80 in normal subjects, 0.70 in Duke's B patients and 1.0 in Duke's C patients. No significant differences were detected between sLe(x) concentrations in sera from normal and neoplastic subjects (p = 0.1). Using an arbitrary cutoff of R = 0.9, the mean disease-free interval in the whole series was 47.4 months for R <0.9 and 126.0 months for R>> or = 0.9 (p = 0.04). The survival time was 76.8 months for patients with R values <0.9 and 156.3 months for patients with R values>> or =0.9 (p = 0.1).

CONCLUSIONS

High serum levels of ganglioside sLe(x) significantly correlate with a favorable prognosis and with a lower occurrence of metastases. It is conceivable that soluble sLe(x) may compete with membrane-bound sLe(x) in the course of interactions between activated endothelium and tumor cells that have reached the circulation.

We report the characterization of a novel series of human endothelial cell lines (designated SGHEC) regarding the expression and release of adhesion molecules and their binding of lymphocytes. SGHEC expressed significant levels of intercellular adhesion molecule-1 (ICAM-1; CD54) which increased after stimulation with tumor necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta), or interferon-gamma (IFN-gamma). Vascular cell adhesion molecule-1 (VCAM-1; CD106) and E-selectin (CD62E) were not detectable on unstimulated SGHEC but substantial levels were expressed after stimulation with either TNF alpha or IL-1 beta but not with IFN-gamma. The increased expression of ICAM-1 and VCAM-1 was evident after 4 h stimulation and was even higher after 24 h; E-selectin was maximal after 4 h and returned almost to basal levels by 24 h. Substantial quantities of immunoreactive ICAM-1 and VCAM-1 also accumulated as soluble material in the supernatants of TNF alpha-stimulated SGHEC (VCAM-1 was substantially higher than ICAM-1), but E-selectin remained below the limits of detection. Various quantitative data suggest that this is a controlled release regulated by cytokine and provide support for a physiological function for these soluble molecules. Primary human lymphocytes and lymphoblastoid cell lines expressing lymphocyte function-associated antigen-1 (LFA-1) bound to SGHEC; this binding increased substantially after activation of either cell type. The binding was inhibited by monoclonal antibodies against LFA-1 and, to a lesser extent, ICAM-1, thus demonstrating the importance of these molecules in the observed binding; neither anti-VCAM-1 nor anti-E-selectin antibodies affected the binding. From these various data, we conclude that LFA-1/ICAM-1 interactions are partially responsible for the binding of lymphocytes to endothelial cells. The SGHEC lines should prove useful in investigating leukocyte-endothelial interactions and the mechanism of release of soluble adhesion molecules.

BACKGROUND

When investigating susceptibility to inflammatory bowel disease (IBD), a multifactorial disorder with a genetic predisposition, polymorphisms of molecules with immunoregulatory function are of potential interest. This is the first time that the polymorphisms of HLA-DR and -DQ, tumour necrosis factor (TNF), E-selectin (CD62E), L-selectin (CD62L), and intercellular adhesion molecule 1 (ICAM-1, CD54) were determined in Estonians, a population with a low IBD incidence rate, and their occurrence investigated in subgroups of a total of 53 IBD patients.

METHODS

The reverse hybridization principle and sequence specific primers were used for HLA genotyping. To analyse the TNF and adhesion molecule polymorphisms, the polymerase chain reaction with subsequent restriction fragment length polymorphism or single-strand conformation polymorphism method was used.

RESULTS

In the subgroup of antineutrophil cytoplasmic antibody (ANCA)-positive ulcerative colitis (UC) patients we found a higher frequency of the TNF2 (20.8% versus 0.0% in ANCA-negative UC patients, P = 0.051) and HLA-DRB1*15 allele (35.4% versus 15.7% in controls; P = 0.004). Of ANCA-positive UC patients 87.5% were carriers of one of these alleles (22.2% among ANCA-negative UC patients (P<0.001, Pc = 0.039) and 51.4% among controls (P = 0.002). Specific typing of HLA-DRB1*15 alleles showed that the HLA-DRB1*1501 allele was responsible for the HLA-DRB1*15 association with ANCA-positive UC. Associations of ICAM-1, E-selectin, or L-selectin polymorphisms with IBD were not found.

CONCLUSIONS

TNF2 and HLA-DRB1*15 alleles were associated with ANCA-positive UC in the investigated population. ANCA might be a useful marker, at least in some ethnic groups, for dividing IBD patients into genetically more homogeneous subgroups.

OBJECTIVE

Tissue degeneration reduces the durability of aortic and pulmonary homograft heart valves. Homograft valves can evoke cellular and humoral immune responses that might be detrimental to the valve tissue. Analyzing explanted homograft valves helps in understanding the different factors that eventually lead to tissue degeneration.

METHODS

A total of 40 homografts was acquired from patients whose grafts had been explanted because of stenosis (n = 22), insufficiency (n = 8), paravalvular leakage (n = 4), other technical problems (n = 4), noncardiac death (n = 1), and stenosis with endocarditis (n = 1). The period of implantation varied from 14 days to 16 years (median, 4 years). Cryopreserved valves (n = 31) were, in the majority, derived from beating-heart donors, whereas the fresh valves were sterilized with antibiotics and stored at 4 degrees C for an average of 32 days. Four unimplanted cryopreserved valves, 1 native aortic valve, and 1 native pulmonary valve were used as references. Analysis included macroscopy, light microscopy with routine hematoxylin and eosin staining (cellularity and tissue structure), and immunohistochemical studies to allow identification of macrophages (CD68) and T lymphocytes (CD3), endothelial cells, leukocyte adhesion molecules (CD54, CD106, and CD62E), and immunoglobulin (IgG) and complement factor (C3) depositions. In situ hybridization for the Y chromosome was performed in 10 cases, with host-donor sex mismatch, to distinguish between host and donor cells. The outcomes of histology and immunohistochemistry were related to clinical factors, such as implantation time and reason for explantation.

RESULTS

In the first year after implantation, a strong reduction in cellularity of the valve tissue was observed, with almost acellular tissues after 1 year. Trilaminar tissue architecture disappeared with the same speed, whereas endothelial cells were almost absent in all explants. Macrophages and T lymphocytes were encountered in 85% and 78% of the leaflets, respectively. Expression of leukocyte adhesion molecules was low in almost all grafts, and IgG and C3 depositions were not increased. Valve tissue cellularity consisted mainly of ingrown host cells when the implantation time exceeded 1 year.

CONCLUSIONS

During the first year of implantation, homograft valves rapidly lose their cellular components and normal tissue architecture. A low-grade inflammatory response was observed, but no convincing evidence of immune-mediated injury was found.

2-(Acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester (NCX-4016) is a nitric oxide (NO)-releasing derivative of aspirin that inhibits cyclooxygenase (COX) activity and releases NO. Acetylation of COX-2 by aspirin activates a transcellular biosynthetic pathway that switches eicosanoid biosynthesis from prostaglandin E(2) to 15-epi-lipoxin (LX)A(4) or aspirin-triggered lipoxin (ATL). Here, we demonstrate that exposure of neutrophil (PMN)/human umbilical vein endothelial cell (HUVEC) cocultures to aspirin and NCX-4016 triggers ATL formation and inhibits cell-to-cell adhesion induced by endotoxin (LPS) and interleukin (IL)-1beta by 70 to 90%. However, although selective and nonselective COX-2 inhibitors (celecoxib, rofecoxib, and naproxen) or N-t-butoxycarbonylmethionine-leucine-phenylalanine (Boc-1), an LXA(4) receptor antagonist, reduced the antiadhesive properties of aspirin by approximately 70%, antiadhesive effects of NCX-4016 were only marginally affected ( approximately 30%) by COX inhibitors and Boc-1, implying that COX-independent mechanisms mediate the antiadhesive properties of NCX-4016. Indeed, NCX-4016 causes a long-lasting (up to 12 h) release of NO and cGMP accumulation in HUVEC. Scavenging NO with 10 mM hemoglobin, in the presence of celecoxib, reduced the antiadhesive properties of NCX-4016 by approximately 80%. Confirming a role for NO, the NO donor diethylenetriamine-NO also inhibited PMN/HUVEC adhesion by approximately 80%. NCX-4016, but not aspirin, decreased DNA binding of nuclear factor-kappaB (NF-kappaB) on gel shift analysis and HUVEC's overexpression of CD54 and CD62E induced by LPS/IL-1beta. Reduction of binding of the two NF-kappaB subunits p50-p50 and p50-p65 was reversed by dithiothreitol, implying S-nitrosylation as mechanism of inhibition. In summary, our results support that ATL and NO are formed at the PMN/HUVEC interface after exposure to NCX-4016 and mediate the antiadhesive properties of this compound.

Circulating endothelial microparticles (EMPs) and progenitor cells (PCs) are biological markers of endothelial function and endogenous repair capacity. The study was aimed to investigate whether COPD patients have an imbalance between EMPs to PCs compared to controls and to evaluate the effect of cigarette smoke on these circulating markers.

Circulating EMPs and PCs were determined by flow cytometry in 27 nonsmokers, 20 smokers and 61 COPD patients with moderate to severe airflow obstruction. We compared total EMPs (CD31+CD42b-), apoptotic if they co-expressed Annexin-V+ or activated if they co-expressed CD62E+, circulating PCs (CD34+CD133+CD45+) and the EMPs/PCs ratio between groups.

COPD patients presented increased levels of total and apoptotic circulating EMPs, and an increased EMPs/PCs ratio, compared with nonsmokers. Women had less circulating PCs than men through all groups and those with COPD showed lower levels of PCs than both control groups. In smokers, circulating EMPs and PCs did not differ from nonsmokers, being the EMPs/PCs ratio in an intermediate position between COPD and nonsmokers.

We conclude that COPD patients present an imbalance between endothelial damage and repair capacity that might explain the frequent concurrence of cardiovascular disorders. Factors related to the disease itself and gender, rather than cigarette smoking, may account for this imbalance.

OBJECTIVE

Stent implantation into atherosclerotic coronary vessels impacts on downstream microvascular function and induces the release of particulate debris and soluble substances, which differs qualitatively and quantitatively between native right coronary arteries (RCAs) and saphenous vein grafts on right coronary arteries (SVG-RCAs). We have now quantified the release of microparticles (MPs) during stent implantation into stable atherosclerotic lesions and compared the release between RCAs and SVG-RCAs.

METHODS

In symptomatic, male patients with stable angina and a stenosis in their RCA or SVG-RCA, respectively (n = 14/14), plaque volume and composition were analyzed using intravascular ultrasound before stent implantation. Coronary aspirate was retrieved during stent implantation with a distal occlusion/aspiration device and divided into particulate debris and plasma. Particulate debris was weighed. Platelet-derived MPs (PMPs) were distinguished by flow cytometry as CD41+, endothelium-derived MPs (EMPs) as CD144+, CD62E+ and CD31+/CD41-, leukocyte-derived MPs as CD45+, and erythrocyte-derived MPs as CD235+.

RESULTS

In patients with comparable plaque volume and composition in RCAs and SVG-RCAs, intracoronary PMPs and EMPs were increased after stent implantation into their RCAs and SVG-RCAs (CD41+: 2729.6 ± 645.6 vs. 4208.7 ± 679.4 and 2355.9 ± 503.9 vs. 3285.8 ± 733.2 nr/µL; CD144+: 451.5 ± 87.9 vs. 861.7 ± 147.0 and 444.6 ± 74.8 vs. 726.5 ± 136.4 nr/µL; CD62E+: 1404.1 ± 247.7 vs. 1844.3 ± 378.6 and 1084.6 ± 211.0 vs. 1783.8 ± 384.3 nr/µL, P < 0.05), but not different between RCAs and SVG-RCAs.

CONCLUSIONS

Stenting in stable atherosclerotic lesions is associated with a substantial release not only of PMPs, but also of EMPs in RCAs and SVG-RCAs. Their release does not differ between RCAs and SVG-RCAs.

BACKGROUND

ClinicalTrials.gov NCT01430884.

BACKGROUND

Inorganic particles, such as drug carriers or contrast agents, are often introduced into the vascular system. Many key components of the in vivo vascular environment include monocyte-endothelial cell interactions, which are important in the initiation of cardiovascular disease. To better understand the effect of particles on vascular function, the present study explored the direct biological effects of particles on human umbilical vein endothelial cells (HUVECs) and monocytes (THP-1 cells). In addition, the integrated effects and possible mechanism of particle-mediated monocyte-endothelial cell interactions were investigated using a coculture model of HUVECs and THP-1 cells. Fe₃O₄ and SiO₂ particles were chosen as the test materials in the present study.

RESULTS

The cell viability data from an MTS assay showed that exposure to Fe₃O₄ or SiO₂ particles at concentrations of 200 μg/mL and above significantly decreased the cell viability of HUVECs, but no significant loss in viability was observed in the THP-1 cells. TEM images indicated that with the accumulation of SiO₂ particles in the cells, the size, structure and morphology of the lysosomes significantly changed in HUVECs, whereas the lysosomes of THP-1 cells were not altered. Our results showed that reactive oxygen species (ROS) generation; the production of interleukin (IL)-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor (TNF)-α and IL-1β; and the expression of CD106, CD62E and tissue factor in HUVECs and monocytes were significantly enhanced to a greater degree in the SiO₂-particle-activated cocultures compared with the individual cell types alone. In contrast, exposure to Fe₃O₄ particles had no impact on the activation of monocytes or endothelial cells in monoculture or coculture. Moreover, using treatment with the supernatants of SiO₂-particle-stimulated monocytes or HUVECs, we found that the enhancement of proinflammatory response by SiO₂ particles was not mediated by soluble factors but was dependent on the direct contact between monocytes and HUVECs. Furthermore, flow cytometry analysis showed that SiO₂ particles could markedly increase CD40L expression in HUVECs. Our data also demonstrated that the stimulation of cocultures with SiO₂ particles strongly enhanced c-Jun NH2-terminal kinase (JNK) phosphorylation and NF-κB activation in both HUVECs and THP-1 cells, whereas the phosphorylation of p38 was not affected.

CONCLUSIONS

Our data demonstrate that SiO₂ particles can significantly augment proinflammatory and procoagulant responses through CD40-CD40L-mediated monocyte-endothelial cell interactions via the JNK/NF-κB pathway, which suggests that cooperative interactions between particles, endothelial cells, and monocytes may trigger or exacerbate cardiovascular dysfunction and disease, such as atherosclerosis and thrombosis. These findings also indicate that the monocyte-endothelial cocultures represent a sensitive in vitro model system to assess the potential toxicity of particles and provide useful information that may help guide the future design and use of inorganic particles in biomedical applications.

Subpopulations of peripheral blood mononuclear cells (PMBCs), known as circulating angiogenic cells (CACs), have been implicated in endothelial repair, angiogenesis and vascular homeostasis. Conversely, microparticles released from endothelial cells, platelets and leucocytes in response to injury or apoptosis are elevated in chronic diseases. We investigated the effect of acute exercise on CAC subpopulations, specifically CD34(+)/VEGFR2(+), CD3(+)/CD31(+), CD14(+)/CD31(+) and CD62E(+) PBMCs and CD62E(+), CD31(+)/CD42b(-) and CD34(+) MPs in men and women. Additionally, we examined angiogenesis-related gene expression in CD34(+), CD31(+) and CD62E(+) PBMCs at baseline and after exercise. Finally, we examined whether acute exercise modulates CD62E(+) PBMC paracrine actions on cultured endothelial cells. Blood samples for CAC and MP analyses were obtained before and after cycling exercise at 70% peak oxygen uptake that elicited an energy expenditure of 600 kcal. Exercise produced a decrease in CD14(+)/CD31(+) PBMCs, whereas CD62E expression on PBMCs increased with exercise. CD34(+)/VEGFR2(+) and CD3(+)/CD31(+) PBMC levels were not altered with exercise. Gene expression analysis revealed a more proangiogenic phenotype in CD62E(+) cells at baseline compared with CD31(+) and CD34(+) cells. Conditioned media from CD62E(+) PBMCs obtained after exercise exerted a proangiogenic influence on human umbilical vein endothelial cells, with increases in genes encoding receptors for growth factors (KDR, FGFR1 and EGFR) and inflammatory mediators (TLR4 and TNFR1). Finally, exercise increased CD62E(+) endothelial MPs in men and increased CD34(+) MPs in women. Our work highlights the potential role of CD62E(+) cells as a novel, exercise-responsive proangiogenic cell population and demonstrates sex-specific exercise-induced changes in circulating MPs.

Increasing evidence links COPD pathogenesis with pulmonary capillary apoptosis. We previously demonstrated that plasma levels of circulating microparticles released from endothelial cells (EMPs) due to apoptosis are elevated in smokers with normal spirometry but low diffusion capacity, that is, with early evidence of lung destruction. We hypothesised that pulmonary capillary apoptosis persists with the development of COPD and assessed its reversibility in healthy smokers and COPD smokers following smoking cessation.

Pulmonary function and high-resolution CT (HRCT) were assessed in 28 non-smokers, 61 healthy smokers and 49 COPD smokers; 17 healthy smokers and 18 COPD smokers quit smoking for 12 months following the baseline visit. Total EMP (CD42b-CD31+), pulmonary capillary EMP (CD42b-CD31+ACE+) and apoptotic EMP (CD42b-CD62E+/CD42b-CD31+) levels were quantified by flow cytometry.

Compared with non-smokers, healthy smokers and COPD smokers had elevated levels of circulating EMPs due to active pulmonary capillary endothelial apoptosis. Levels remained elevated over 12 months in healthy smokers and COPD smokers who continued smoking, but returned to non-smoker levels in healthy smokers who quit. In contrast, levels remained significantly abnormal in COPD smokers who quit.

Pulmonary capillary apoptosis is reversible in healthy smokers who quit, but continues to play a role in COPD pathogenesis in smokers who progressed to airflow obstruction despite smoking cessation.

NCT00974064; NCT01776398.

Mesangial cells are one of the three major cell types of the kidney glomerulus that provide physical support for the glomerular capillary lumen of the kidney. Loss of mesangial cells due to pathologic conditions, such as glomerulonephritis and diabetic nephropathy, can impair renal function. Mesenchymal stem cells (MSC) are attractive candidates for kidney repair therapy since they can enhance recovery and protect against kidney failure. MSC can differentiate into mesangial cells in vivo. We have investigated the ability of MSC to differentiate into mesangial cells in vitro; they were co-cultured with oxidant-injured mesangial cells before being analysed by flow cytometry and for contractility. MSC co-cultured with injured mesangial cells had a mesangial cell-like morphology and contracted in response to angiotensin II. They expressed CD54(-) CD62E(+) in direct contrast to the CD54(+) CD62E(-) of pure MSC. In conclusion, MSC can differentiate into mesangial cells in vitro when co-cultured with injured mesangial cells.

OBJECTIVE

Degenerative aortic valve stenosis (AVS) is independently associated with endothelial dysfunction and increased levels of circulating endothelium-derived microparticles (EMPs) as a marker of compromised endothelial integrity. The aim of this study was to investigate whether therapy for severe AVS by transcatheter aortic valve implantation (TAVI) improves endothelial function and decreases EMPs.

RESULTS

Fifty-six patients with indication for TAVI due to symptomatic severe AVS were prospectively enrolled. Brachial wall shear stress (WSS), endothelial function and circulating microparticles (MPs) were measured before and three months following TAVI. Endothelial function was assessed as flow-mediated dilation (FMD) using ultrasound. MP subpopulations were discriminated by flow cytometry according to the expression of established surface antigens: CD31+/CD41-, CD144+ and CD62E+ as EMPs and CD41+ as platelet-derived MPs (PMPs). In patients with severe AVS, decreased brachial WSS was an independent predictor of low FMD. At three-month follow-up after TAVI, WSS and FMD increased along with decreased levels of EMPs as compared to pre TAVI. Decrease of CD31+/CD41-, CD144+ and CD62E+ EMP levels correlated with the increase of FMD.

CONCLUSIONS

Therapy for AVS by TAVI was associated with improved endothelial function and integrity indicating beneficial effects of TAVI on systemic arterial function.

Lysis of aortic endothelial cells (EC) by neutrophils from spontaneously hypertensive rats (SHR) was investigated using a nonradioactive cytotoxicity assay. Interleukin-1-activated EC, but not unstimulated EC, were effective target cells for lysis by SHR neutrophils. Supernatants from activated neutrophil did not exert a cytotoxic effect on EC. Inhibitors of reactive oxygen species did not affect the cytotoxicity of neutrophils on EC. In contrast, inhibitors of serine protease and elastase markedly inhibited the cytotoxicity of neutrophils on EC. Antibodies against the endothelial cell surface ligands ICAM-1 (CD54) and E-selectin (CD62E) inhibited the adhesion and cytotoxicity of activated neutrophils on EC. The cytotoxicity of neutrophils required direct cell-to-cell contact because separating them with a microporous membrane abrogated the neutrophil-mediated cytotoxic activity. These results demonstrate that SHR neutrophils possess potent cytotoxicity against cytokine-activated EC. Neutrophil-mediated damage of EC could contribute to organ damage in hypertension under conditions of local or systemic activation of neutrophils.

BACKGROUND

The hemodynamic vascular consequences of implanting left ventricular assist devices (LVADs) have not been studied in detail. We investigated the effect of LVAD implantation compared with heart transplant (HTx) on microvascular and macrovascular function in patients with end-stage heart failure and evaluated whether microparticles may play a role in LVAD-related endothelial dysfunction.

METHODS

Vascular function was assessed in patients with end-stage heart failure awaiting HTx, patients who had undergone implantation of a continuous-flow centrifugal LVAD, and patients who had already received a HTx. Macrovascular function was measured by flow-mediated vasodilation (FMD) using high-resolution ultrasound of the brachial artery. Microvascular function was assessed in the forearm during reactive hyperemia using laser Doppler perfusion imaging and pulsed wave Doppler. Age-matched patients without heart failure and without coronary artery disease (CAD) (healthy control subjects) and patients with stable CAD served as control subjects. Circulating red blood cell (CD253(+)), leukocyte (CD45(+)), platelet (CD31(+)/CD41(+)), and endothelial cell (CD31(+)/CD41(-), CD62e(+), CD144(+)) microparticles were determined by flow cytometry and free hemoglobin by enzyme-linked immunosorbent assay.

RESULTS

FMD and microvascular function were significantly impaired in patients with end-stage heart failure compared with healthy control subjects and patients with stable CAD. LVAD implantation led to recovery of microvascular function, but not FMD. In parallel, increased free hemoglobin was observed along with red and white cell microparticles and endothelial and platelet microparticles. This finding indicates destruction of blood cells with release of hemoglobin and activation of endothelial cells. HTx and LVAD implantation led to similar improvements in microvascular function. FMD increased and microparticle levels decreased in patients with HTx, whereas shear stress during reactive hyperemia was similar in patients with LVADs and patients with HTx.

CONCLUSIONS

Our data suggest that LVAD support leads to significant improvements in microvascular perfusion and hemodynamics. However, destruction of blood cells may contribute to residual endothelial dysfunction potentially by increasing nitric oxide scavenging capacity.

BACKGROUND

Activation of cytoskeleton regulator Rho-kinase during inflammatory stimulations plays a major role in cellular dysfunction and apoptosis. Because endothelial dysfunction may be influenced by increased circulating membrane microparticles (MPs), we hypothesized that inhibition of Rho-kinase can prevent interferon-α (IFN-α)-induced endothelial cell (EC) apoptosis and that protective effects of Rho-kinase inhibition are facilitated by prevention of F-actin rearrangement.

METHODS

In this study, Lewis rats were subjected to an intraperitoneal injection of IFN-α or IFN-α + Y-27632. FCM was performed to analyze circulating endothelial MPs (EMPs) from the blood samples of these animals by detecting the expression of CD144, CD62E, CD31, CD51, and CD54 on EMPs. IFN-α-induced pulmonary injury was assessed by measurement of lung wet-to-dry weight ratios and measurement of alveolar wall thickness. Human pulmonary microvascular ECs (HPMECs) were cultured with IFN-α or EMPs to elucidate the probable mechanisms of the release of EMPs.

RESULTS

Injection of IFN-α resulted in much higher levels of CD144 EMPs, CD62E EMPs, CD31 EMPs, CD51 EMPs, and CD54 EMPs. Pulmonary injury was also observed after injection of IFN-α. Furthermore, IFN-α induced F-actin rearrangement and apoptosis of HPMECs in vitro, and the Toll-like receptor 4/MyD88 and nuclear factor-κB pathways and EMPs per se played important roles in this process.

CONCLUSIONS

The results demonstrate that increased Rho-kinase activity causes the release of EMPs and cellular apoptosis. Moreover, HPMEC apoptosis that resulted from EMP stimulation indicates that EMPs can be considered as a potential target to regulate the rearrangement of cytoskeleton during endothelial cell apoptosis.

BACKGROUND

Accelerating atherosclerosis in type 2 diabetes mellitus (T2DM) patients may relate to imbalance between pattern of microparticles (MPs), which are frequently involved in repair of vasculature, tissue injury, inflammation and thrombosis. The aim of the study was to investigate the pattern of circulating MPs in T2DM patients with asymptomatic atherosclerosis.

METHODS

A total of 103 patients with T2DM (54 subjects without documented coronary atherosclerosis and 49 patients with angiographic evidence of asymptomatic coronary atherosclerosis) who were underwent a contrast-enhanced multispiral computed tomography angiography and 35 healthy volunteers were enrolled in the study. To determine circulating biomarkers, blood samples were collected at baseline. MPs were labelled and characterized by flow cytometry.

RESULTS

There were no significant differences between healthy volunteers and T2DM patients in circulating numbers of MPs labelled as CD41a+, CD64+, CD144+, CD144+/CD31+, Annexin V+, CD144+/annexin V+ and CD144+/CD31+/annexin V+. However, lower number of MPs with immune phenotypes CD62E+, CD105E+ and higher numbers of CD31+/annexin V+ MPs were reported in T2DM patients when compared with healthy volunteers. Therefore, we found an increased level of circulating CD41a+ MPs, CD144+/CD31+ MPs, CD31+/annexin V+ MPs, and decreased level of CD62E+ MPs in T2DM patients with asymptomatic coronary atherosclerosis in comparison with those who had no asymptomatic atherosclerosis. Using multivariate log regression analysis, BMI (odds ratio [OR] = 1.04, p = 0.001), LDL-C (OR = 1.05, p = 0.046), hs-CRP (OR = 1.07, p = 0.044), osteoprotegerin (OR = 1.07, p = 0.026), CD62E+ MPs (OR = 1.07, p = 0.001) and CD31+/annexin V+ MPs (OR = 1.12, p = 0.003) were determined independent predictive factors of asymptomatic atherosclerosis in T2DM patients.

CONCLUSIONS

Circulating levels of MP originated from apoptotic endothelial cell-derived were significantly increased in diabetic patients as compared with normal subjects, but level of activated endothelial cell-derived MPs was lower than in healthy volunteers. Among T2DM patients, an increased level of CD31+/annexin V+ MPs and decreased CD62E+ MPs were significantly associated with asymptomatic atherosclerosis.

Expression of the cell adhesion molecule (CAM), Sialyl Lewis X (CD15s) correlates with cancer metastasis, while expression of E-selectin (CD62E) is stimulated by TNF-α. CD15s/CD62E interaction plays a key role in the homing process of circulating leukocytes. We investigated the heterophilic interaction of CD15s and CD62E in brain metastasis-related cancer cell adhesion. CD15s and CD62E were characterised in human brain endothelium (hCMEC/D3), primary non-small cell lung cancer (NSCLC) (COR-L105 and A549) and metastatic NSCLC (SEBTA-001 and NCI-H1299) using immunocytochemistry, Western blotting, flow cytometry and immunohistochemistry in human brain tissue sections. TNF-α (25 pg/mL) stimulated extracellular expression of CD62E while adhesion assays, under both static and physiological flow live-cell conditions, explored the effect of CD15s-mAb immunoblocking on adhesion of cancer cell-brain endothelium. CD15s was faintly expressed on hCMEC/D3, while high levels were observed on primary NSCLC cells with expression highest on metastatic NSCLC cells (p < 0.001). CD62E was highly expressed on hCMEC/D3 cells activated with TNF-α, with lower levels on primary and metastatic NSCLC cells. CD15s and CD62E were expressed on lung metastatic brain biopsies. CD15s/CD62E interaction was localised at adhesion sites of cancer cell-brain endothelium. CD15s immunoblocking significantly decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (p < 0.001), highlighting the role of CD15s-CD62E interaction in brain metastasis.

Design of tissue-engineered cell-loaded device involves cells seeding onto scaffolds in vitro, allowing them to settle and grow before in vivo transplantation. Interaction between scaffold and cells is important in the development of desired tissues. The present study aimed to investigate the effect of cell-polymer interactions on cell morphology and expression of surface markers of osteogenic MBA-15 cells cultured on various bioresorbable polymers. In this study, we used various polymers: poly(L-lactic acid) (PLLA), poly(DL-lactic acid) (PDLLA), poly(L-lactic-glycolic acid) (PLGA), and poly(DL-lactide-glycolide acid) PDLGA1 and PDLGA2. Expression of integrinalpha-M (CD11b), selectin-E (CD62E), and PECAM-1 (CD31), important in cell-cell and cell-matrix interactions, were quantified by flow-cytometry analysis. Cells grown on PDLGA1 films demonstrated fivefold increase in CD62E expression and two-folds increase in CD11b expression. None of the polymers affected the levels of CD31. Identified differential effect of polymers on the expression of cell-adhesion molecules by osteoprogenitors in vitro might help to choose optimal parameters for successful engraftment of cell-loaded constructs.