CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ) inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC). We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106) expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d) resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood.

Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56(+) cells grew rapidly, a population of CD15(+) cells emerged, partly from CD56(+) cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56(+) and CD15(+) cells shared osteogenic and chondrogenic abilities, while CD56(+) cells presented a myogenic capacity and CD15(+) cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.

Transplantation of growth factor-mobilized peripheral blood progenitor cells (PBPC) is widely used in the treatment of several neoplastic diseases. While in PBPC harvests the presence of several accessory immune and tumor cells has been documented, that of stromal cells has not been reported. In the present study, we investigated for the presence of stromal cells in growth factor-mobilized PBPC harvests from breast cancer patients. Low-density cells from PBCP harvests in culture gave rise to an adherent layer containing fibroblast-like and large flat round cells. These cells express positive immunofluorescence staining for collagen I, collagen III, fibronectin, VCAM-1 (CD106), ICAM-1 (CD54) and mesenchymal antigens recognized by monoclonal antibodies, SH2 and SH3. PBPC-derived stromal cells do not express antigens CD34, CD45 and CD14. Stromal cells were detected in the PBPC harvests of 11/14 patients (median 0.63%; range 0.02-2.32) and their concentration correlates with the number of CD34+ cells in PBPC.

Bone formation in mammals requires continuous production of osteoblasts throughout life. A common molecular marker for all osteogenic mesenchymal progenitors has not been identified. Here, by lineage-tracing experiments in fetal or postnatal mice, we discover that Gli1+ cells progressively produce osteoblasts in all skeletal sites. Most notably, in postnatal growing mice, the Gli1+ cells residing immediately beneath the growth plate, termed here "metaphyseal mesenchymal progenitors" (MMPs), are essential for cancellous bone formation. Besides osteoblasts, MMPs also give rise to bone marrow adipocytes and stromal cells in vivo. RNA-seq reveals that MMPs express a number of marker genes previously assigned to mesenchymal stem/progenitor cells, including CD146/Mcam, CD44, CD106/Vcam1, Pdgfra, and Lepr. Genetic disruption of Hh signaling impairs proliferation and osteoblast differentiation of MMPs. Removal of β-catenin causes MMPs to favor adipogenesis, resulting in osteopenia coupled with increased marrow adiposity. Finally, postnatal Gli1+ cells contribute to both chondrocytes and osteoblasts during bone fracture healing. Thus Gli1 marks mesenchymal progenitors responsible for both normal bone formation and fracture repair.

BACKGROUND

Embryonic stem cells possess the ability to differentiate into endothelium. The ability to produce large volumes of endothelium from embryonic stem cells could provide a potential therapeutic modality for vascular injury. We describe an approach that selects endothelial cells using magnetic beads that may be used therapeutically to treat arterial injury.

RESULTS

Large numbers of endothelial cells (ECs) with high purity were produced using Sca-1+ cells isolated with magnetic beads from predifferentiated embryonic stem cells (ESCs) cultured in alpha-MEM containing 10 ng/mL VEGF165 for a minimum of 21 days (esEC). The transcription regulator histone deacetylase (HDAC3) was essential for VEGF-induced EC differentiation. Immunofluorescence or fluorescence-activated cell sorter (FACS) analysis revealed that esECs expressed a full range of EC lineage-specific markers including CD31, CD106, CD144, Flk-1, Flt-1, and von Willebrand factor (vWF). FACS analysis confirmed that 99% of esECs were CD31-positive and 75% vWF-positive. Furthermore, almost all cells were positive for DiI-acLDL uptake. When matrigel containing esECs was subcutaneously implanted into mice, various vessel-like structures were observed indicating their endothelial cell like phenotype. In keeping with this, when esECs infected with adenovirus-LacZ were injected into denuded femoral arteries of mice, they were found to form a neo-endothelium that covered the injured areas (86%+/-13.6%), which resulted in a 73% decrease in neointimal area 2 weeks after injury.

CONCLUSIONS

We conclude that Sca-1+ cells can differentiate into functional ECs via activation of HDAC3, accelerating re-endothelialization of injured arteries and reducing neointima formation.

Current sources of mesenchymal cells, including bone marrow, fat and muscle, all require invasive procurement procedures, and provide relatively low frequencies of progenitors. Here, we describe the non-invasive isolation, and characterization, of a rich source of mesenchymal progenitor cells, which we call human umbilical cord perivascular cells (HUCPVCs). HUCPVCs show a similar immunological phenotype to bone marrow-derived mesenchymal stromal cells (BM-MSCs), since they are non-alloreactive, exhibit immunosuppression, and significantly reduce lymphocyte activation, in vitro. They present a non-hematopoietic myofibroblastic mesenchymal phenotype (CD45-, CD34-, CD105+, CD73+, CD90+, CD44+, CD106+, 3G5+, CD146+); with a 1:300 frequency at harvest, a short-doubling time, and a clonogenic frequency of >1:3 in culture. Furthermore, in addition to robust quinti-potential differentiation capacity in vitro, HUCPVCs have been shown to contribute to both musculo-skeletal and dermal wound healing in vivo.

The consequences of internalization of Staphylococcus aureus by HUVEC with respect to their adhesiveness for human monocytes and granulocytes were investigated. Viable and UV-killed, but not heat-killed, S. aureus were internalized by HUVEC, which required participation of the endothelial cytoskeleton. S. aureus-infected HUVEC displayed increased surface expression of CD106 (VCAM-1), CD54 (ICAM-1), and MHC I molecules. Expression of CD62P (P-selectin), CD62E (E-selectin), CD31 (PECAM-1), and CD102 (ICAM-2) was not affected. Concomitantly, these HUVEC expressed a time- and inoculum size-dependent hyperadhesiveness for monocytes and granulocytes. Monocyte adhesion reached maximal levels (approximately 60% adhesion) 23 h after the initial 1 h period of infection of HUVEC with about 50 bacteria per single HUVEC. To induce maximal (approximately 20%) adhesion of granulocytes, five times higher concentrations of HUVEC-infecting bacteria were required. Using the appropriate mAb, granulocyte adhesion to S. aureus-infected HUVEC was shown to be entirely mediated by the beta2 (CD11/CD18) integrins. Monocyte adhesion to these HUVEC was largely (approximately 70%) dependent on both CD11a/CD18 (LFA-1) and CD49d/CD29 (VLA-4). This demonstrates that infection of HUVEC with S. aureus potentiates CD11/CD18-mediated granulocyte adhesion and shifts the mechanism of monocyte adhesion from being completely CD11/CD18 dependent to one that also utilizes the VLA-4/VCAM-1 dependent pathway. Together, these findings indicate that in response to internalization of S. aureus, vascular endothelial cells may initiate recruitment of monocytes and granulocytes, which may be an important initial event in the pathogenesis of endovascular diseases.

Thymic nurse cells are known to interact with T cells and play a role in their functional maturation. However, the role of nurse cells in B cell maturation and differentiation is less well established, especially at extralymphoid sites. To address this issue, nurse-like cell clones from bone marrow and synovial tissue of patients with RA (RA-NLC) were established and characterized. RA-NLC constitutively expressed CD29, CD49c, CD54 (ICAM-1), CD106 (VCAM-1), CD157 (BST-1), and class I MHC molecules, and secreted IL-6, IL-7, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). Bone marrow-derived and synovial RA-NLC differed in that the former secreted IL-7 and expressed a greater density of CD157 constitutively and after stimulation with IFNgamma, whereas the latter secreted G-CSF and more IL-6. Stimulation of both bone marrow and synovial RA-NLC induced expression of CD40 and class II MHC, but not CD154 (CD40L) or CD35. RA-NLC rescued peripheral B cells from spontaneous apoptosis and promoted survival of B cells for>> 4 wk. B cell survival was blocked by antibodies to CD106 or CD157. RA-NLC also increased Ig production from B cells. After long-term culture (4-6 wk) with RA-NLC, but not alone or with fibroblasts, outgrowth of B cells was observed. All B cell lines derived from these cultures had been transformed by EBV, although the RA-NLC themselves were not infected with EBV. Precursor frequency analysis indicated that approximately 1 in 12,500 peripheral B cells could give rise to these EBV-transformed B cell lines upon coculture with RA-NLC. These results indicate that RA-NLC from bone marrow and synovium have the capacity to rescue B cells from spontaneous apoptosis, facilitate Ig production, and promote the outgrowth of EBV-transformed B lymphoblastoid cells. These findings suggest that RA-NLC may play a role in the local and systemic hyperreactivity of B cells characteristic of rheumatoid arthritis.

OBJECTIVE

Contradictory results have been reported regarding vasculogenesis in systemic sclerosis (SSc). Our aim was to investigate bone marrow-derived circulating endothelial precursors (EPCs) and activated circulating endothelial cells (CECs) in SSc patients.

METHODS

Peripheral blood from consecutive patients with SSc hospitalised for systemic follow-up was analysed and compared with blood from patients with active refractory rheumatoid arthritis (RA) and osteoarthritis (OA). EPCs were quantified by cell sorting and flow cytometry and were identified as circulating CD34+CD133+ cells. Activated CECs were defined as CD105+CD62+CD105+CD102+CD105+CD106+ cells.

RESULTS

Patients with SSc had higher putative EPC levels than OA patients, but lower levels than RA patients. In SSc patients, EPC levels increased with European disease activity score. Activated CEC levels were high in SSc patients and RA patients, but not correlated with EPC levels.

CONCLUSIONS

These results together and previous data suggest that EPCs may be recruited during active vascular disease but that the sustained ischaemic conditions of SSc may eventually lead to EPCs depletion.

Mesenchymal stem cells (MSCs) have been successfully isolated from a broad range of adult, fetal, and other nonembryonic tissues. Fetal lung has been identified as a rich source of MSCs. However, the biological characteristics and differentiation potential of fetal lung MSCs remain to be explored. In this study, we established a series of methods for isolation and expansion of fetal lung MSCs. These MSCs could withstand more than 40 passages without obvious decline in proliferation ability, significant changes in morphology, and expression of cell markers. Flow cytometric analysis showed that fetal lung MSCs expressed CD13, CD29, CD44, CD90, CD105, CD166, and HLA-ABC, but not CD14, CD31, CD34, CD38, CD41a, CD42b, CD45, CD49d, CD61, CD106, CD133, and HLA-DR. Cell cycle analysis revealed that when the MSCs reached their log phase of growth, more than 90% of the cells were in G0/G1 phase while the proportion of cells in S phase and G2/M phase were about 5.56% and 2.08% cells, respectively. These MSCs could differentiate into neural cells in addition to their mesenchymal differentiation potential. Our data suggest that the fetal lung MSC population is an alternative source of stem cells for cell-based therapy of neurological defects or mesenchymal-originating diseases.

OBJECTIVE

To assess the feasibility of seeding adipose-derived stem cells (ADSCs) onto bladder acellular matrix grafts (BAMGs) for bladder reconstruction in a rabbit model.

METHODS

Autologous ADSCs were isolated, expanded and identified by flow cytometry. In the experimental group, ADSCs were seeded onto BAMGS for reconstructing bladder defects in 12 male rabbits. Unseeded BAMGs were used for bladder reconstruction in the control group of 12 rabbits. Cystography was performed at 4, 12 and 24 weeks after grafts implantation. Following cystography, the animals were killed and grafts were harvested; H&E and immunohistochemical staining were performed with cytokeratin AE1/AE3, smooth muscle alpha-actin and S-100 markers.

RESULTS

Flow cytometry demonstrated that the ADSCs expressed CD90, CD44, CD105, CD166 and CD34, but not CD45 or CD106. The cells demonstrated good biocompatibility with BAMGs. At 24 weeks, in the experimental group, the reconstructed bladders reached a mean volume of 94.68 +/- 3.31% of the pre-cystectomy bladder capacity. Complete regeneration of smooth muscle and nerve tissue was evident. Regenerated SMCs, urothelium and nerve cells stained positively for alpha-smooth muscle actin, AE1/AE3 and S-100. In the control group, the mean bladder volume was 69.33 +/- 5.05% of the pre-cystectomy volume; histologically, the control group was characterized by multi-layered urothelium without evidence for organized muscle or nerve tissue.

CONCLUSIONS

These data demonstrate that seeding ADSCs onto BAMGs promote regeneration of smooth muscle and nervous tissue regeneration in a rabbit model. This compound graft was more suitable for bladder reconstruction than BAMG alone.

Expression of E-selectin (ELAM-1, CD62E) on human umbilical vein endothelial cells significantly increased 30 min postinfection with the flavivirus West Nile virus (WNV), was maximal by 2 h postinfection, and declined to baseline levels within 24 h. Expression of ICAM-1 (CD54) and VCAM-1 (CD106) was significantly increased by 2 h and maximal at 4 h after infection. P-selectin (CD62P) expression was unaffected by WNV. Upregulation occurred earlier than that caused by tumor necrosis factor alpha (TNF-alpha) or interleukin 1 (IL-1) and could not be inhibited by neutralizing TNF-alpha, IL-1alpha, or alpha/beta interferon (IFN-alpha/beta) antibodies, suggesting a direct, virus-mediated phenomenon. TNF-alpha significantly enhanced WNV-induced increases in E-selectin, P-selectin, ICAM-1, and VCAM-1 expression, while IFN-gamma enhanced WNV-induced ICAM-1 expression. In contrast, IL-4 abrogated WNV-induced E-selectin expression increases but acted in synergy with WNV to increase P-selectin and VCAM-1 expression. WNV increased the expression of class I and II major histocompatibility complex antigens (MHC-I and MHC-II, respectively) at 24 and 72 h, respectively. IFN-gamma, TNF-alpha, or IL-1 acted in synergy with WNV to produce greater increases in MHC-I expression than WNV or cytokines alone, while IFN-alpha/beta or IL-4 had no effect. MHC-II induction in cytokine-treated, WNV-infected cells was similar to that caused by cytokines alone. Neutralizing IFN-alpha/beta antibody inhibited WNV-induced MHC-I expression by 30% at 24 h and by 100% by 72 h. The differential kinetics of modulation suggest sequential adhesion of leukocyte subpopulations to infected endothelial cells, which may be important in initial viral spread in vivo.

Airway epithelium may actively participate in inflammatory responses, such as occur in asthma. The presence and regulation of surface molecules on the airway epithelium, however, is incompletely understood. We have determined the phenotype of the human bronchial epithelial cell line BEAS-2B by flow cytometry. We confirmed previous observations that human bronchial epithelial cells constitutively express CD29, CD44, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54 (ICAM-1), CD61, and HLA class 1. BEAS-2B cells were also found to constitutively express CD9, CD13, CD15, CD15s, CD23, CD33, CD36, CD40, CD41b, CD42b, CD48, CD50, CD71, and CD102 (ICAM-2). Culture of BEAS-2B cells with tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta (1 ng/ml) was found to enhance intercellular adhesion molecule-1 (ICAM-1) expression (several fold) and induce de novo CD106 [vascular cell adhesion molecule-1 (VCAM-1)] expression. TNF-alpha or IL-1beta did not change the expression of CD9, CD13, CD16, CD23, CD29, CD31, CD32, CD35, CD45, CD61, or CD64 in BEAS-2B cells. IL-4 (1 ng/ml) also induced expression of VCAM-1 (1.5-fold) but not ICAM- expression while interferon-gamma (1 ng/ml) enhanced only ICAM-1 expression (2-fold). Maximal VCAM-1 expression was obtained with the combination of TNF-alpha and IL-4 (8-fold). Using Northern blot hybridization analysis, ICAM-1 and VCAM-1 mRNA was detected in BEAS-2B cells stimulated with cytokines. VCAM-1 on stimulated BEAS-2B was functionally active as determined by adhesion of purified eosinophils and blockade with specific antibodies. Primary isolates of bronchial epithelial cells produced detectable levels of VCAM-1 protein and mRNA as detected by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. These results suggest that cytokine activation induces expression of ICAM-1 and VCAM-1 on airway epithelium, an event which may influence leukocyte infiltration and activation.

Epidemiologic studies have shown an association between exposure to ambient particulate air pollution <10 microm in diameter (PM(10)) and increased cardiovascular morbidity and mortality. We previously showed that PM(10) exposure causes progression of atherosclerosis in coronary arteries. We postulate that the recruitment of monocytes from the circulation into atherosclerotic lesions is a key step in this PM(10)-induced acceleration of atherosclerosis. The study objective was to quantify the recruitment of circulating monocytes into vessel walls and the progression of atherosclerotic plaques induced by exposure to PM(10). Female Watanabe heritable hyperlipidemic rabbits, which naturally develop systemic atherosclerosis, were exposed to PM(10) (EHC-93) or vehicle by intratracheal instillation twice a week for 4 wk. Monocytes, labeled with 5-bromo-2'-deoxyuridine (BrdU) in donors, were transfused to recipient rabbits as whole blood, and the recruitment of BrdU-labeled cells into vessel walls and plaques in recipients was measured by quantitative histological methodology. Exposure to PM(10) caused progression of atherosclerotic lesions in thoracic and abdominal aorta. It also decreased circulating monocyte counts, decreased circulating monocytes expressing high levels of CD31 (platelet endothelial cell adhesion molecule-1) and CD49d (very late antigen-4 alpha-chain), and increased expression of CD54 (ICAM-1) and CD106 (VCAM-1) in plaques. Exposure to PM(10) increased the number of BrdU-labeled monocytes adherent to endothelium over plaques and increased the migration of BrdU-labeled monocytes into plaques and smooth muscle underneath plaques. We conclude that exposure to ambient air pollution particles promotes the recruitment of circulating monocytes into atherosclerotic plaques and speculate that this is a critically important step in the PM(10)-induced progression of atherosclerosis.

Embryonic mesenchymal stem cells (eMSCs) were first derived from human embryonic stem cells (hESCs) overexpressing green fluorescence protein (GFP). They expressed CD29, CD44, CD73, CD105, CD166 and nestin, but not CD34, CD45, CD106 SSEA-4 or Oct3/4. Twenty million eMSCs in 1 mL of phosphate-buffered saline (PBS) were injected into the femoral veins of spontaneously hypertensive rats after transient middle cerebral artery occlusion. The migration and differentiation of the eMSCs in the ischemic brain were analyzed. The results revealed that eMSCs migrated to the infarction region and differentiated into neurons, which were positive for beta-tubulin III, microtubule-associated protein 2 (MAP2), HuC, neurofilament and human nuclear antibody, and to vascular endothelial cells, which were positive for von Willebrand factor (vWF). The transplanted cells survived in the infarction region for at least 4 weeks. Adhesive removal function significantly improved in the first week after cell transplantation, and rotarod motor function significantly improved starting from the second week. The infarction volume in the eMSC group was significantly smaller than that in the PBS control group at 4 weeks after infusion. The results of this study show that when administered intravenously, eMSCs differentiated into neuronal and endothelial cells, reduced the infarction volume, and improved behavioral functional outcome significantly in transient focal cerebral ischemia.

OBJECTIVE

The aim of the current study was to determine levels of circulating endothelial cell adhesion molecules during preeclampsia and to assess their predictive value as diagnostic markers for the early identification of pregnant women at risk of developing preeclampsia.

METHODS

Plasma samples were obtained from women with preeclampsia; the syndrome of hemolysis, elevated liver enzymes, and low platelets; uncomplicated pregnancy-induced hypertension; and women with normal pregnancy. In addition, longitudinal plasma profiles of pregnant women were randomly collected to determine individual profiles of circulating endothelial cell adhesion molecules. A sandwich enzyme-linked immunosorbent assay technique was used to quantitate concentrations of soluble intercellular adhesion molecule-1 (CD54), vascular cell adhesion molecule-1 (CD106), E-selectin (CD62E), platelet endothelial cell adhesion molecule (CD31), and P-selectin (CD62P).

RESULTS

Plasma levels of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, and platelet endothelial cell adhesion molecule-1 were significantly elevated in women with preeclampsia compared with healthy control pregnant women. Longitudinal analysis of soluble plasma intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 levels during pregnancy revealed that these molecules (1) show little variation in healthy pregnant women, (2) do not vary during normal pregnancy, and (3) are significantly elevated in women with preeclampsia and the syndrome of hemolysis, elevated liver enzymes, and low platelets compared with control pregnant women and those with uncomplicated pregnancy-induced hypertension. Analysis of soluble intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 levels in longitudinal profiles of pregnant women identified significantly elevated levels of these molecules in the plasma of preeclampsia-prone women 3 to 15 weeks before the onset of clinical symptoms.

CONCLUSIONS

Elevated soluble intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 measurements during pregnancy can be considered as major risk factors. Elevated levels of these substances in the plasma of pregnant women with preeclampsia support the concept of a primary endothelial cell involvement in the pathogenesis of preeclampsia. Although currently based on a limited database, significantly elevated levels of soluble intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in the plasma of otherwise healthy pregnant women suggest a very high predictive value of these molecules for the earliest identification of women at risk of developing preeclampsia.

Asthma is characterized by appearance of eosinophils in the airway. Eosinophils purified from the airway 48 h after segmental antigen challenge are described as exhibiting greater adhesion to albumin-coated surfaces via an unidentified beta2 integrin and increased expression of alphaMbeta2 (CD11b/18) compared with purified blood eosinophils. We have investigated the determinants of this hyperadhesive phenotype. Airway eosinophils exhibited increased reactivity with the CBRM1/5 anti-alphaM activation-sensitive antibody as well as enhanced adhesion to VCAM-1 (CD106) and diverse ligands, including albumin, ICAM-1 (CD54), fibrinogen, and vitronectin. Purified blood eosinophils did not adhere to the latter diverse ligands. Enhanced adhesion of airway eosinophils was blocked by anti-alphaMbeta2. Podosomes, structures implicated in cell movement and proteolysis of matrix proteins, were larger and more common on airway eosinophils adherent to VCAM-1 when compared with blood eosinophils. Incubation of blood eosinophils with IL-5 replicated the phenotype of airway eosinophils. That is, IL-5 enhanced recognition of alphaM by CBRM1/5; stimulated alphaMbeta2-mediated adhesion to VCAM-1, albumin, ICAM-1, fibrinogen, and vitronectin; and increased podosome formation on VCAM-1. Thus, the hyperadhesion of airway eosinophils after antigen challenge is mediated by upregulated and activated alphaMbeta2.

According to the prevailing paradigm, neutrophils are short-lived cells that undergo spontaneous apoptosis within 24 hours of their release from the bone marrow. However, neutrophil survival can be significantly prolonged within inflamed tissue by cytokines, inflammatory mediators, and hypoxia. During screening experiments aimed at identifying the effect of the adhesive microenvironment on neutrophil survival, we found that VCAM-1 (CD106) was able to delay both spontaneous and Fas-induced apoptosis. VCAM-1-mediated survival was as efficient as that induced by the cytokine IFN-beta and provided an additive, increased delay in apoptosis when given in combination with IFN-beta. VCAM-1 delivered its antiapoptotic effect through binding the integrin alpha9beta1. The alpha9beta1 signaling pathway shares significant features with the IFN-beta survival signaling pathway, requiring PI3 kinase, NF-kappaB activation, as well as de novo protein synthesis, but the kinetics of NF-kappaB activation by VCAM-1 were slower and more sustained compared with IFN-beta. This study demonstrates a novel functional role for alpha9beta1 in neutrophil biology and suggests that adhesive signaling pathways provide an important extrinsic checkpoint for the resolution of inflammatory responses in tissues.

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.

A number of studies have identified a role for plasminogen activator inhibitor-1 (PAI-1) in regulating angiogenesis, although results from these investigations have been controversial. Among key cellular components of an angiogenic vessel are endothelial cells (ECs), which are known to express several components of the fibrinolytic system, including PAI-1. Thus, alterations in expression of this protein may have direct effects on cell functions involved in vascular development. In this study, ECs were isolated from sections of murine arterial trees from wild-type and PAI-1-deficient mice, and low passage (passages 3-4) homogeneous subpopulations of these cells were obtained by immunomagnetic absorption to antibodies against CD105/CD106. The homogeneity of these cells was further assessed by immunohistochemistry and quantitative real-time reverse transcription-PCR analysis of a number of EC markers. Comparative analyses of EC proliferation (one event associated with angiogenesis) in wild-type and PAI-1-deficient ECs demonstrated enhanced rates of cell growth for PAI-1-deficient cells relative to wild-type cells. Additional studies demonstrated similar levels of both vascular endothelial growth factor (VEGF) mRNA and protein and enhanced levels of VEGF receptor-1 (Flt-1) mRNA in PAI-1-deficient cells relative to wild-type cells. Immunohistochemical analyses indicated that phosphorylation of Akt was also enhanced in PAI-1-deficient cells, implicating VEGF-induced cell signaling alterations in PAI-1-deficient cells, the result of which may contribute to alterations in cell proliferation.

Tumor angiogenesis and immune response have in common to be cell recognition mechanisms, which are based on specific adhesion molecules and dependent on nitric oxide (NO(•)). The aim of the present study is to deepen the mechanisms of angiogenesis and inflammation regulation by NO(•) to find out the molecular regulation processes that govern endothelial cell permeability and leukocyte transmigration. Effects of NO(•), either exogenous or produced in hypoxic conditions, were studied on microvascular endothelial cells from skin and lymph node because of their strong involvement in melanoma progression. We found that NO(•) down-regulation of pseudo-vessel formation was linked to a decrease in endothelial cell ability to adhere to each other which can be explain, in part, by the inhibition of PECAM-1/CD31 expression. On the other hand, NO(•) was shown to be able to decrease leukocyte adhesion on an endothelial monolayer, performed either in static or in rolling conditions, and to modulate differentially CD34, ICAM-1/CD54, ICAM-2/CD102 and VCAM-1/CD106 expression. In conclusion, during angiogenesis and leukocyte recruitment, NO(•) regulates cell interactions by controlling adhesion molecule expression and subsequently cell adhesion. Moreover, each endothelial cell type presents its own organospecific response to NO(•), reflecting the functions of the tissue they originate from.

Stable mixed hematopoietic chimerism has been consistently established in dogs who were mildly immunosuppressed by 200 cGy of total body irradiation (TBI) before undergoing dog leukocyte antigen (DLA)-identical bone marrow (BM) transplantation and who received a brief course of immunosuppression with mycophenolate mofetil (28 days) and cyclosporine (35 days) after transplantation. However, when TBI was reduced from 200 to 100 cGy, grafts were nearly uniformly rejected within 3-12 weeks. Here, we asked whether stable engraftment could be accomplished after a suboptimal dose of 100 cGy TBI with host immunosuppression enhanced by donor-derived mesenchymal stromal cells (MSCs) given after transplantation. MSCs were cultured from BM cells and evaluated in vitro for antigen expression. They showed profound immunosuppressive properties in mixed lymphocyte reactions (MLRs) in a cell dose-dependent manner not restricted by DLA. MSC and lymphocyte contact was not required, indicating that immunosuppression was mediated by soluble factors. Prostaglandin E2 was increased in culture supernatant when MSCs were cocultured in MLRs. The addition of indomethacin restored lymphocyte proliferation in cultures containing MSCs. MSCs expressed CD10, CD13, CD29, CD44, CD73/SH-3, CD90/Thy-1, and CD106/VCAM-1. For in vivo studies, MSCs were injected on the day of BM grafting and on day 35, the day of discontinuation of posttransplantation cyclosporine. MSCs derived from the respective BM donors failed to avert BM graft rejection in 4 dogs who received DLA-identical grafts after nonmyeloablative conditioning with 100 cGy TBI in a time course not significantly different from that of control dogs not given MSCs. Although the MSCs displayed in vitro characteristics similar to those reported for MSCs from other species, their immunosuppressive qualities failed to sustain stable BM engraftment in vivo in this canine model.

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.