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Introduction High-grade serous ovarian cancer (HGSC) is the most common histological subtype of ovarian cancer and accounts for most ovarian cancer-related deaths. Most HGSCs are diagnosed at a late stage, [...], The molecular mechanism by which cancer-associated fibroblasts (CAFs) confer chemoresistance In ovarian cancer Is poorly understood. The purpose of the present study was to evaluate the roles of CAFs in modulating tumor vasculature, chemoresistance, and disease progression. Here, we found that CAFs upregulated the lipoma-preferred partner (LPP) gene in microvascular endothelial cells (MECs) and that LPP expression levels in intratumoral MECs correlated with survival and chemoresistance in patients with ovarian cancer. Mechanistically, LPP increased focal adhesion and stress fiber formation to promote endothelial cell motility and permeability. siRNA- mediated LPP silencing in ovarian tumor-bearing mice improved paclitaxel delivery to cancer cells by decreasing intratumoral microvessel leakiness. Further studies showed that CAFs regulate endothelial LPP via a calcium-dependent signaling pathway involving microfibrillar-associated protein 5 (MFAP5), focal adhesion kinase (FAK), ERK, and LPP. Thus, our findings suggest that targeting endothelial LPP enhances the efficacy of chemotherapy in ovarian cancer. Our data highlight the importance of CAF-endothelial cell crosstalk signaling in cancer chemoresistance and demonstrate the improved efficacy of using LPP-targeting siRNA in combination with cytotoxic drugs.

T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and [...]

Introduction Cancer growth strictly depends on the expansion of the host vasculature, which not only supplies oxygen and nutrients to the tumor tissue, but also provides cancer cells with the [...], While tumor blood vessels share many characteristics with normal vasculature, they also exhibit morphological and functional aberrancies. For example, the neural adhesion molecule L1, which mediates neurite outgrowth, fasciculation, and pathfinding, is expressed on tumor vasculature. Here, using an orthotopic mouse model of pancreatic carcinoma, we evaluated L1 functionality in cancer vessels. Tumor-bearing mice specifically lacking L1 in endothelial cells or treated with anti-L1 antibodies exhibited decreased angiogenesis and improved vascular stabilization, leading to reduced tumor growth and metastasis. In line with these dramatic effects of L1 on tumor vasculature, the ectopic expression of L1 in cultured endothelial cells (ECs) promoted phenotypical and functional alterations, including proliferation, migration, tubulogenesis, enhanced vascular permeability, and endothelial-to-mesenchymal transition. L1 induced global changes in the EC transcriptome, altering several regulatory networks that underlie endothelial pathophysiology, including JAK/STAT-mediated pathways. In particular, L1 induced IL-6-mediated STAT3 phosphorylation, and inhibition of the IL-6/JAK/STAT signaling axis prevented L1-induced EC proliferation and migration. Evaluation of patient samples revealed that, compared with that in noncancerous tissue, L1 expression is specifically enhanced in blood vessels of human pancreatic carcinomas and in vessels of other tumor types. Together, these data indicate that endothelial L1 orchestrates multiple cancer vessel functions and represents a potential target for tumor vascular- specific therapies.

Most patients who die from cancer succumb to treatment-refractory advanced metastatic progression. Although the early stages of tumor metastasis result in the formation of clinically silent micrometastatic foci, its later stages primarily reflect the progressive, organ-destructive growth of already advanced metastases. Early-stage metastasis is regulated by multiple factors within tumor cells as well as by the tumor microenvironment (TME). In contrast, the molecular determinants that control advanced metastatic progression remain essentially uncharacterized, precluding the development of therapies targeted against it. Here we show that the TME, functioning in part through platelet endothelial cell adhesion molecule 1 (PECAM-1), drives advanced metastatic progression and is essential for progression through its preterminal end stage. PECAM1-KO and chimeric mice revealed that its metastasis-promoting effects are mediated specifically through vascular endothelial cell (VEC) PECAM-1. Anti-PECAM-1 mAb therapy suppresses both end-stage metastatic progression and tumor-induced cachexia in tumor-bearing mice. It reduces proliferation, but not angiogenesis or apoptosis, within advanced tumor metastases. Because its antimetastatic effects are mediated by binding to VEC rather than to tumor cells, antiPECAM-1 mAb appears to act independently of tumor type. A modified 3D coculture assay showed that anti--PECAM-1 mAb inhibits the proliferation of PECAM-1--negative tumor cells by altering the concentrations of secreted factors. Our studies indicate that a complex interplay between elements of the TME and advanced tumor metastases directs end-stage metastatic progression. They also suggest that some therapeutic interventions may target late-stage metastases specifically, mAb-based targeting of PECAM-1 represents a TME-targeted therapeutic approach that suppresses the end stages of metastatic progression, until now a refractory clinical entity. metastasis | endothelium | tumor microenvironment doi/ 10.1073/pnas.1004654107

Platelet endothelial cell adhesion molecule-1 (PECAM-1) has been implicated in endothelial cell motility during angiogenesis. Although there is evidence that SHP-2 plays a role in PECAM-1-dependent cell motility, the molecular basis of the activity of SHP-2 in this process has not been defined. To investigate the requirement of SHP-2 in PECAM-1-dependent cell motility, studies were done in which various constructs of SHP-2 were expressed in cell transfectants expressing PECAM-1. We observed that the levels of PECAM-1 tyrosine phosphorylation and SHP-2 association with PECAM-1 were significantly increased in cells expressing a phosphatase-inactive SHP-2 mutant, suggesting that the level of PECAM-1 tyrosine phosphorylation, and thus SHP-2 binding are regulated in part by bound, catalytically active SHP-2. We subsequently found that expression of PECAM-1 stimulated wound-induced migration and the formation of filopodia (a morphological feature of motile cells). These activities were associated with increased mitogen-activated protein kinase (MAPK) activation and the dephosphorylation of paxillin (an event implicated in the activation of MAPK). The phosphatase-inactive SHP-2 mutant, however, suppressed these PECAM-1-dependent phenomena, whereas the activity of PECAM-1 expressing cells was not altered by expression of wild-type SHP-2 or SHP-2 in which the scaffold/adaptor function had been disabled. Pharmacological inhibition of SHP-2 phosphatase activity also suppressed PECAM-1-dependent motility. Furthermore, PECAM-1 expression also stimulates tube formation, but none of the SHP-2 constructs affected this process. These findings therefore suggest a model for the involvement of SHP-2 in PECAM-1-dependent motility in which SHP-2, recruited by its interaction with PECAM-1, targets paxillin to ultimately activate the MAPK pathway and downstream events required for cell motility. mitogen-activated protein kinase; angiogenesis; leukocyte transendothelial migration doi: 10.1152/ajpcell.00436.2009.

The specificity of interactions between neurons is believed to be mediated by diverse cell adhesion molecules, including members of the cadherin superfamily. Whereas mechanisms of classical cadherin adhesion have been studied extensively, much less is known about the related protocadherins (Pcdhs), which together make up the majority of the superfamily. Here we use quantitative cell aggregation assays and biochemical analyses to characterize cis and trans interactions among the 22-member [gamma]-Pcdh family, which have been shown to be critical for the control of synaptogenesis and neuronal survival. We show that [gamma]-Pcdh isoforms engage in trans interactions that are strictly homophilic. In contrast to classical cadherins, [gamma]-Pcdh interactions are only partially [Ca.sup.2+]-dependent, and their specificity is mediated through the second and third extracellular cadherin (EC) domains (EC2 and EC3), rather than through EC1. The [gamma]-Pcdhs also interact both covalently and noncovalently in the cis-orientation to form multimers both in vitro and in vivo. In contrast to [gamma]-Pcdh trans interactions, cis interactions are highly promiscuous, with no isoform specificity. We present data supporting a model in which [gamma]-Pcdh cistetramers represent the unit of their adhesive trans interactions. Unrestricted tetramerization in cis, coupled with strictly homophilic interactions in trans, predicts that the 22 [gamma]-Pcdhs could form 234,256 distinct adhesive interfaces. Given the demonstrated role of the [gamma]-Pcdhs in synaptogenesis, our data have important implications for the molecular control of neuronal specificity. cadherin | calcium-dependent | synaptogenesis | recognition | trafficking doi/ 10.1073/pnas.1004526107

Cell adhesion molecules are key players in transsynaptic communication, precisely coordinating presynaptic differentiation with postsynaptic specialization. At glutamatergic synapses, their retrograde signaling has been proposed to control presynaptic vesicle clustering at active zones. However, how the different types of cell adhesion molecules act together during this decisive step of synapse maturation is largely unexplored. Using a knockout approach, we show that two synapUc adhesion systems, N-cadherin and neuroligin-1, cooperate to control vesicle clustering at nascent synapses. Live cell imaging and fluorescence recovery after photobleaching experiments at individual synaptic boutons revealed a strong impairment of vesicle accumulation in the absence of N-cadherin, whereas the formation of active zones was largely unaffected. Strikingly, also the clustering of synaptic vesicles triggered by neuroligin-1 overexpression required the presence of N-cadherin in cultured neurons. Mechanistically, we found that N-cadherin acts by postsynaptically accumulating neuroligin-1 and activating its function via the scaffolding molecule S-SCAM, leading, in turn, to presynaptic vesicle clustering. A similar cooperation of N-cadherin and neuroligin-1 was observed in immature CA3 pyramidal neurons in an organotypic hippocampal network. Moreover, at mature synapses, N-cadherin was required for the increase in release probability and miniature EPSC frequency induced by expressed neuroligin-1. This cooperation of two cell adhesion systems provides a mechanism for coupling bidirectional synapse maturation mediated by neuroligin-1 to cell type recognition processes mediated by classical cadherins. synapse maturation | synaptic adhesion molecules | vesicle clustering doi/ 10.1073/pnas.0914233107

Formation of neural circuits depends on stable contacts between neuronal processes, mediated by interaction of cell adhesion molecules, including N-cadherin. In the present study, we found that activity-dependent dendrite arborization specifically requires Ncadherin--mediated extracellular neuron-neuron interaction, because the enhancerffent did not occur for neurons cultured in isolation or plated on an astrocyte monolayer and was abolished by a recombinant soluble N-cadherin ectodomain. Furthermore, depolarization elevated the level of membrane-associated cadherin/catenin complexes and surface N-cadherin. Importantly, surface N-cadherin elevation is specifically required for the maintenance of nascent dendrite arbors. Through loss--and gain-of-function approaches, we showed that N-cadherin-mediated dendrite growth requires association of the cadherin/catenin complex with the actin cytoskeleton. In summary, these results identify a previously unexplored and specific function for activity-induced, N-cadherin-mediated neuron-neuron contacts in the maintenance of dendrite arbors. cell adhesion molecule | catenin | actin www.pnas.org/cgi/doi/10.1073/pnas.1003480107

Neuronal growth cones are highly motile structures that tip developing neurites and explore their surroundings before axodendritic contact and synaptogenesis. However, the membrane proteins organizing these processes remain insufficiently understood. Here we identify that the synaptic cell adhesion molecule 1 (SynCAM 1), an immunoglobulin superfamily member, is already expressed in developing neurons and localizes to their growth cones. Upon interaction of growth cones with target neurites, SynCAM 1 rapidly assembles at these contacts to form stable adhesive clusters. Synaptic markers can also be detected at these sites. Addressing the functions of SynCAM 1 in growth cones preceding contact, we determine that it is required and sufficient to restrict the number of active filopodia. Further, SynCAM 1 negatively regulates the morphological complexity of migrating growth cones. Focal adhesion kinase, a binding partner of SynCAM 1, is implicated in its morphogenetic activities. These results reveal that SynCAM 1 acts in developing neurons to shape migrating growth cones and contributes to the adhesive differentiation of their axo-dendritic contacts. CADM | focal adhesion kinase | growth cone | synaptic adhesion | synaptogenesis doi/ 10.1073/pnas.0911798107

Atherogenesis involves activation of NF-[kappa]B in endothelial cells by fluid shear stress. Because this pathway involves integrins, we investigated the involvement of focal adhesion kinase (FAK). We found that FAK was not required for flow-stimulated translocation of the p65 NF-[kappa]B subunit to the nucleus but was essential for phosphorylation of p65 on serine 536 and induction of ICAM-1, an NF-[kappa]B-dependent gene. NF-[kappa]B activation by TNF-[alpha] or hydrogen peroxide was FAK independent. Events upstream of NF-[kappa]B, including integrin activation, Rac activation, reactive oxygen production, and degradation of I[kappa]B, were FAK independent. FAK therefore regulates NF-[kappa]B phosphorylation and transcriptional activity in response to flow by a novel mechanism. atherosclerosis; fluid shear stress; integrin signaling; mechanotransduction doi: 10.1152/ajpcell.00226.2009

Many cell--cell adhesive events are mediated by the dimerization of cadherin proteins presented on apposing cell surfaces. Cadherin-mediated processes play a central role in the sorting of cells into separate tissues in vivo, but in vitro assays aimed at mimicking this behavior have yielded inconclusive results. In some cases, cells that express different cadherins exhibit homotypic cell sorting, forming separate cell aggregates, whereas in other cases, intermixed aggregates are formed. A third pattern is observed for mixtures of cells expressing either N- or E-cadherin, which form distinct homotypic aggregates that adhere to one another through a heterotypic interface. The molecular basis of cadherin-mediated cell patterning phenomena is poorly understood, in part because the relationship between cellular adhesive specificity and intermolecular binding free energies has not been established. To clarify this issue, we have measured the dimerization affinities of N-cadherin and E-cadherin. These proteins are similar in sequence and structure, yet are able to mediate homotypic cell patterning behavior in a variety of tissues. N-cadherin is found to form homodimers with higher affinity than does E-cadherin and, unexpectedly, the N/E-cadherin heterophilic binding affinity is intermediate in strength between the 2 homophilic affinities. We can account for observed cell aggregation behaviors by using a theoretical framework that establishes a connection between molecular affinities and cell-cell adhesive specificity. Our results illustrate how graded differences between different homophilic and heterophilic cadherin dimerizaton affinities can result in homotypic cell patterning and, more generally, show how proteins that are closely related can, nevertheless, be responsible for highly specific cellular adhesive behavior. binding affinities | cadherins | cell adhesion | differential adhesion hypothesis | surface plasmon resonance

In many organs, integrins and cadherins are partly regulated by Hox genes, but their interactions in airway morphogenesis and congenital lung diseases are unknown. We previously showed that the Hox protein HoxB5 is abnormally increased in bronchopulmonary sequestration (BPS) and congenital cystic adenomatoid malformation (CCAM), congenital lung lesions with abnormal airway branching. We now report on [[alpha].sub.2]-, [[alpha].sub.3]-, and [[beta].sub.1]-integrin and E-cadherin expression in normal human lung and in BPS and CCAM tissue previously shown to have abnormal HoxB5 expression and on the relationship of cell adhesion molecule expression to Hoxb5 regulation, [[alpha].sub.2]-, [[alpha].sub.3]-, and [[beta].sub.1]-integrins and E-cadherin expression in normal human lung and BPS and CCAM were evaluated using Western blot and immunohistochemistry. Fetal mouse lung fibroblasts with Hoxb5-specific siRNA downregulation were evaluated for [[alpha].sub.2]-integrin protein levels by Western blot. Compared with normal human lung, a previously undetected [[alpha].sub.2]-integrin isoform potentially lacking essential cytoplasmic sequences was significantly increased in BPS and CCAM, and [[alpha].sub.2]-integrin spatial and cellular expression was more intense. E-cadherin protein levels were also significantly increased, whereas [[alpha].sub.3] increased in CCAM compared with canalicular, but not with alveolar, stage lung. [[beta].sub.1]-integrin levels were unchanged. We conclude that in BPS and CCAM, altered [[alpha].sub.2]-integrin cytoplasmic signaling contributes to abnormal cellular behavior in these lung lesions. Aberrant cell adhesion molecule and Hox protein regulation are likely part of the mechanism involved in the development of BPS and CCAM. [[alpha].sub.2]-integrin; [[alpha].sub.3]-integrin; E-cadherin; Hox proteins

E-cadherin plays a key role at adherens junctions between epithelial cells, but the mechanisms controlling its assembly, maintenance, and dissociation from junctions remain poorly understood. In particular, it is not known to what extent the number of E-cadherins engaged at junctions is regulated by endocytosis, or by dissociation of adhesive bonds and redistribution within the membrane from a pool of diffusive cadherins. To determine whether cadherin levels at mature junctions are regulated by endocytosis or dissociation and membrane diffusion, the dynamics of E-cadherin were quantitatively analyzed by a new approach combining 2-photon fluorescence recovery after photobleaching (FRAP) and fast 3D wide-field fluorescence microscopy. Image analysis of fluorescence recovery indicates that most E-cadherin did not diffuse in the membrane along mature junctions, but followed a first order turn-over process that was rate-limited by endocytosis. In confluent cultures of MCF7 or MDCK cells, stably expressed EGFP-E-cadherin was rapidly recycled with spatially uniform kinetics (50 s in MCF7 and 4 min in MDCK). In addition, when endocytosis was pharmacologically blocked by dynasore or MiTMAB, no fluorescence recovery was observed, suggesting that no endocytosis-independent membrane redistribution was occurring. Our data show that membrane redistribution of E-cadherin molecules engaged in mature junctions requires endocytosis and subsequent exocytosis, and lead to the notion that E-cadherins engaged at junctions do not directly revert to free membrane diffusion. Our results point to the possibility that a direct mechanical coupling between endocytosis efficiency and cadherin-mediated forces at junctions could help to regulate intercellular adhesion and locally stabilize epithelia. diffusion | fluorescence recovery after photobleaching

Endothelial lipase (EL) is a novel phospholipase that determines plasma high-density lipoprotein cholesterol (HDL-C) levels. We have investigated the role of HDL-C in lung allergic inflammation by using EL knockout (EL-KO) mice that are high in HDL-C. EL-KO and wild-type control mice were sensitized and challenged with ovalbumin to evoke eosinophilic inflammation in the lung. EL was expressed in epithelial cells, alveolar type II cells, and endothelial cells in the lung, and its expression was upregulated during inflammation. Concomitant with attenuated hyperresponsiveness of the airway smooth muscles, the number of eosinophils in bronchoalveolar lavage and the expression of VCAM-1 were lower in EL-KO mice than in control mice. HDL reduced cytokine-induced VCAM-1 expression in cultured endothelial cells. When plasma HDL levels were decreased to similar levels in both mouse groups by adenovirus-mediated overexpression of EL, however, eosinophil infiltration was still lower in EL-KO mice. In vitro adhesion assays revealed that EL expression on the cell surface promoted the interaction of eosinophils through the ligand-binding function of EL. In summary, targeted inactivation of EL attenuated allergic inflammation in the lung, and the protective effects in EL-KO mice were associated with high plasma HDL levels, downregulation of VCAM-1, and loss of the direct ligand-binding function of EL. Thus EL is a novel modulator of the progression of allergic asthma. high-density lipoprotein; cholesterol; adhesion molecule; allergy; endothelium

Patten recognition receptors, which recognize pathogens or components of injured ceils (danger), trigger activation of the innate immune system. Whether and how the host distinguishes between danger- versus pathogen-associated molecular patterns remains unresolved. We report that CD24-deficient mice exhibit increased susceptibility to danger- but not pathogen-associated molecular patterns. CD24 associates with high mobility group box 1, heat shock protein 70, and heat shock protein 90; negatively regulates their stimulatory activity; and inhibits nuclear factor [kappa]B (NF-[kappa]B) activation. This occurs at least in part through CD24 association with Siglec-10 in humans or Siglec-G in mice. Our results reveal that the CD24-Siglec G pathway protects the host against a lethal response to pathological cell death and discriminates danger- versus pathogen-associated molecular patterns.

Hexosamine biosynthetic pathway (HBP) accounts for some cardiovascular adverse effects of hyperglycemia. We investigated whether the HBP inhibitor azaserine protects against hyperglycemia-induced endothelial damage dependently of HBP. Human endothelial cells isolated from umbilical veins were exposed either to a high (30.5 mmol/l) or low concentration of glucose (5.5 mmol/l) for 4 days, followed by a stimulation with TNF-[alpha] (1 ng/ml, 24 h). The blockade of the rate-limiting enzyme glutamine:fructose-6-phosphate amido-transferase inhibited HBP flux and oxidative stress (generation of superoxide and peroxynitrite) under the hyperglycemic condition and prevented the synergistic stimulation of VCAM-1 and ICAM-1 expression by hyperglycemia and TNF-[alpha]. In the cells cultured under a low-glucose condition when no increased HBP flux occurred, azaserine enhanced the manganese-superoxide dismutase (MnSOD) protein level and also inhibited the oxidative stress and the expression of VCAM-1 and ICAM-1 in response to TNF-[alpha]. Moreover, the polyphenol resveratrol inhibited the oxidative stress and adhesion molecule expression and did not decrease the HBP flux under the hyperglycemia condition. In addition, in isolated rat aortas exposed to hyperglycemic buffer for 5 h when no significant HBP flux occurred, azaserine upregulated the MnSOD protein level and prevented decreased endothelium-dependent relaxations to acetylcholine. In conclusion, hyperglycemia independently increases oxidative stress and HBP flux, amplifies endothelial inflammation, and impairs endothelial function mainly through oxidative stress and not the HBP pathway. Azaserine protects against hyperglycemic endothelial damage through its antioxidant effect independently of inhibiting HBP pathway. adhesion molecules; endothelium; glucose; O-linked acetylglusamine; superoxide

Down syndrome cell adhesion molecule (DSCAM) is required for axon guidance and dendrite arborization. How DSCAM functions in vertebrates is not well understood. Here we show that DSCAM is expressed on commissural axons and interacts with Netrin-1, a prototypical guidance cue for commissural axons. The knockdown of DSCAM by specific siRNA or blockage of DSCAM signaling by overexpression of a mutant lacking its intracellular domain inhibits netrin-induced axon outgrowth and commissural axon turning in vitro. SiRNA-mediated knockdown of DSCAM in ovo causes defects in commissural axon projection and pathfinding. In transfected cells, DSCAM by itself, in the absence of DCC, is capable of mediating netrin signaling in activating phosphorylation of Fyn and Pak1. These findings demonstrate an essential role of vertebrate DSCAM in axon guidance, indicating that DSCAM functions as a receptor of netrin-1. Our data suggest previously unexpected complexity in receptors that mediate vertebrate netrin signaling. neurite outgrowth | commissural axon guidance | neuronal guidance receptor | signal transduction

p120-catenin regulates epithelial cadherin stability and has been suggested to function as a tumor suppressor. In this study, we used anchorage-independent growth (AIG), a classical in vitro tumorigenicity assay, to examine the role of p120 in a different context, namely oncogene-mediated tumorigenesis. Surprisingly, p120 ablation by short hairpin RNA completely blocked AIG induced by both Rac1 and Src. This role for p120 was traced to its activity in suppression of the RhoA-ROCK pathway, which appears to be essential for AIG. Remarkably, the AIG block associated with p120 ablation was completely reversed by inhibition of the downstream RhoA effector ROCK. Harvey-Ras (H-Ras)-induced AIG was also dependent on suppression of the ROCK cascade but was p120 independent because its action on the pathway occurred downstream of p120. The data suggest that p120 modulates oncogenic signaling pathways important for AIG. Although H-Ras bypasses p120, a unifying theme for all three oncogenes is the requirement to suppress ROCK, which may act as a gatekeeper for the transition to anchorage independence.

Activation of the CD40 receptor by its cognate ligand, CD 154, results in interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) production and increased intercellular adhesion molecule-1 (ICAM-1) expression in proximal tubule cells (PTCs). The independent role of these two proinflammatory chemokines, IL-8 and MCP-1, in inciting an inflammatory response in PTCs was explored. Exposure of primary cultures of human renal PTCs to recombinant IL-8 and MCP-1 resulted in increased ICAM-1 expression measured by quantitative real-time PCR, but confirmed only for IL-8 by immunoblot. The mechanism of action of IL-8 was explored in further detail. Immunohistochemistry identified both the CXCR-1 and CXCR-2 receptors, confirmed by RT-PCR, immunoprecipitation, immunoblot, and FACS analysis. IL-8 increased ICAM-1 expression only via the CXCR-1 receptor, which in turn resulted in activation of the p38 mitogen-activated protein kinase (MAPK) pathway; neither the extracellular signal-related kinase (ERK) 1/2 MAPK pathway nor the stress-activated protein kinase (SAPK)/c-Jun NH2 terminal kinase (JNK) pathway was involved. CD154/CD40-mediated ICAM-1 upregulation was not affected by preincubation of monolayers with the CXCR-1 blocking antibody, indicating that ICAM-1 expression occurs independent of CD154-mediated IL-8 production. Coincubation of monolayers with both CD154 and IL-8 resulted in a greater ICAM-1 response than either compound alone. We conclude that in human renal PTCs, IL-8 upregulates ICAM-1 production by engaging the CXCR-1 receptor and p38 MAPK signaling pathway. This cascade of events is independent of CD40/CD154-mediated IL-8 stimulation and ICAM-1 production and serves to amplify the inflammatory response. chemokines; IL-8 receptor; mitogen-activated protein kinases; interstitial inflammation; kidney cells

This study demonstrates that in malignant melanoma, elevated levels of nuclear [beta]-catenin in both primary tumors and metastases correlate with reduced expression of a marker of proliferation and with improved survival, in contrast to colorectal cancer. The reduction in proliferation observed in vivo is recapitulated in B16 murine melanoma cells and in human melanoma cell lines cultured in vitro with either WNT3A or small-molecule activators of [beta]-catenin signaling. Consistent with these results, B16 melanoma cells expressing WNT3A also exhibit decreased tumor size and decreased metastasis when implanted into mice. Genome-wide transcriptional profiling reveals that WNT3A up-regulates genes implicated in melanocyte differentiation, several of which are down-regulated with melanoma progression. These findings suggest that WNT3A can mediate transcriptional changes in melanoma cells in a manner reminiscent of the known role of Wnt/[beta]-catenin signaling in normal melanocyte development, thereby altering melanoma cell fate to one that may be less proliferative and potentially less aggressive. Our results may explain the observed loss of nuclear [beta]-catenin with melanoma progression in human tumors, which could reflect a dysregulation of cellular differentiation through a loss of homeostatic Wnt/[beta]-catenin signaling. differentiation | prognosis | metastasis | WNT5A | B16 model | microarray

Selectins are adhesion molecules that resist large tensile forces applied by hydrodynamic forces to leukocytes binding to vessel walls. In crystals, the liganded (high-affinity) and unliganded (low-affinity) conformations differ in orientation between their tandem lectin and EGF domains. I examine how tensile force exerted on a selectin-ligand complex in vivo could favor the more extended, high-affinity conformation. AIIostery is transmitted from the EGF-lectin domain interface to the ligand-binding interface on the lectin domain, 30 [Angstrom] away. Trp-1 of the lectin domain and the long axis of the EGF domain form an L-shaped prybar that is welded together by hydrogen bonds to the Trp-1 [alpha]-amino group. Pivoting of the prybar induced by force demolishes an interface between the Trp-1 side chain and the lectin domain at a switch1 region. These changes are transmitted by rigid body movement of the switch2 region to rearrangements in the switch3 region at the ligand binding site. Another switch region corresponds to a single residue in the EGF domain with large effects on ligand binding and rolling adhesion. Allostery in selectins, and the alignment of tensile force on a selectin-ligand complex with the transition pathway for conformational change, explain much of the structural basis for selectin mechanochemistry.

The selectins are cell adhesion proteins that must resist applied forces to mediate leukocyte tethering and rolling along the endothelium and have 2 conformational states. Selectin-ligand bond dissociation increases only modestly with applied force, and exhibits catch bond behavior in a low-force regime where bond lifetimes counterintuitively increase with increasing force. Both allosteric and sliding-rebinding models have emerged to explain catch bonds. Here, we introduce a large residue into a cleft that opens within the lectin domain to stabilize the more extended, high-affinity selectin conformation. This mutation stabilizes the high-affinity state, but surprisingly makes rolling less stable. The position of the mutation in the lectin domain provides evidence for an allosteric pathway through the lectin domain, connecting changes at the lectin-EGF interface to the distal binding interface. cell rolling | mechanochemistry | vasculature

Localization of presynaptic components to synaptic sites is critical for hippocampal synapse formation. Cell adhesion--regulated signaling is important for synaptic development and function, but little is known about differentiation of the presynaptic compartment. In this study, we describe a pathway that promotes presynaptic development involving p120catenin (p120ctn), the cytoplasmic tyrosine kinase Fer, the protein phosphatase SHP-2, and [beta]-catenin. Presynaptic Fer depletion prevents localization of active zone constituents and synaptic vesicles and inhibits excitatory synapse formation and synaptic transmission. Depletion of p120ctn or SHP-2 similarly disrupts synaptic vesicle localization with active SHP-2, restoring synapse formation in the absence of Fer. Fer or SHP-2 depletion results in elevated tyrosine phosphorylation of [beta]-catenin. [beta]-Catenin overexpression restores normal synaptic vesicle localization in the absence of Fer or SHP-2. Our results indicate that a presynaptic signaling pathway through p120ctn, Fer, SHP-2, and [beta]-catenin promotes excitatory synapse development and function.

The L1 family of single-pass transmembrane cell adhesion molecules (L1CAMs) is conserved from Caenorhabditis elegans and Drosophila to vertebrates and is required for axon guidance, neurite outgrowth, and maintenance of neuronal positions. The extracellular region of L1CAMs mediates cell adhesion via interactions with diverse cell-surface and extracellular matrix proteins. In contrast, less is known regarding the function of the intracellular domains in the L1CAM cytoplasmic tail. Previously, we identified a role of the C. elegans L1CAM homolog, SAX-7, in maintaining neuronal and axonal positioning. Here, we demonstrate that this function is dependent on three conserved motifs that reside in the SAX-7 cytoplasmic tail: (1) the FERM-binding motif, (2) the ankyrin-binding domain, and (3) the PDZ-binding motif. Furthermore, we provide molecular and genetic evidence that UNC-44 ankyrin and STN-2 [gamma]-syntrophin bind SAX-7 via the respective ankyrin-binding and PDZ-binding motifs to regulate SAX-7 function in maintaining neuronal positioning.

Ischemiareperfusion (IR) injury is a major insult to postcapillary venules. We hypothesized that IR increases postcapillary venular hydraulic conductivity and that IR-mediated changes in hydraulic conductivity result from temporally and mechanistically separate processes. A microcannulation technique was used to determine hydraulic conductivity ([L.sub.p]) in rat mesenteric postcapillary venules serially throughout ischemia (45 min) and reperfusion (5 h) induced by superior mesenteric artery occlusion and release. Mesenteric IR resulted in a biphasic increase in [L.sup.p]. White blood cell (WBC) adhesion slowly increased with maximal adhesion corresponding to the second peak (P < 0.005). After IR, tissue was harvested for RT-PCR analysis of ICAM-1, E-selectin, and P-selectin mRNA. Intercellular adhesion molecule-1 (ICAM-1) mRNA in the gut showed the most significant upregulation. Quantitative real-time PCR revealed that ICAM-1 mRNA was upregulated 60-fold in the gut. An ICAM-1 antibody was therefore used to determine the effect of WBC adhesion on [L.sub.p] during IR. ICAM-1 inhibition attenuated [L.sub.p] during the first peak and completely blocked the second peak (P < 0.005). When rats were fed a tungsten diet to inhibit xanthine oxidase and then underwent IR, [L.sub.p] was dramatically attenuated during the first peak and mildly decreased the second peak (P < 0.005). Inhibition of xanthine oxidase by oxypurinol decreased [L.sub.p] during IR by over 60% (P < 0.002). Tempol, a superoxide dismutase mimetic, decreased [L.sub.p] during IR by over 30% (P < 0.01). We conclude that IR induces a biphasic increase in postcapillary hydraulic conductivity. Reactive oxygen species impact both the first transient peak and the sustained second peak. However, the second peak is also dependent on WBC-endothelial cell adhesion. These serial measurements of postcapillary hydraulic conductivity may lead the way for optimal timing of pharmaceutical therapies in IR injury. microvascular permeability; reactive oxygen species; intercellular adhesion molecule-1

We examined the effects of supernatants of culture media of MDA-MB-231 and MCF-7 cells on the expression of adhesion molecules on human lymphatic endothelial cells (LECs) and evaluated whether the overexpression of adhesion molecules facilitated the attachment of carcinoma cells to LECs. The 48-h stimulation of MDA-MB-231, but not MCF-7, supernatant produced a significant expression of ICAM-1 on human LECs but little or no expression of E-selectin. Chemical treatment with dialyzed substances of intercellular adhesion molecule-1; sentinel lymph node

Two key characteristics of the inflammatory response are the recruitment of leukocytes to inflamed tissue as well as changes in vessel permeability. We explored the relationship between these two processes using intravital confocal microscopy in cremasters of anesthetized (65 mg/kg Nembutal ip) mice. We provide direct evidence that intercellular adhesion molecule-1 (ICAM-1) links leukocyte-endothelial cell interactions and changes in solute permeability ([P.sub.s]). Importantly, we show that arterioles, not just venules, respond to proinflammatory stimuli, thus contributing to microvascular exchange. We identified two independent, ICAM-1-mediated pathways regulating [P.sub.s]. Under control conditions in wild-type (WT) mice, there is a constitutive PKC-dependent pathway ([P.sub.s] = 1.0 [+ or -] 0.10 and 2.2 [+ or -] 0.46 x [10.sup.-6] cm/s in arterioles and venules, respectively), which was significantly reduced in ICAM-1 knockout (KO) mice ([P.sub.s] = 0.54 [+ or -] 0.07 and 0.77 [+ or -] 0.11 x [10.sub.-6] cm/s). The PKC inhibitor hisindolylmaleimid 1 (1 [micro]mol/l in 0.01% DMSO) decreased [P.sub.s] in WT mice to levels similar to those in ICAM-1 KO mice. Likewise, a PKC activator (phorbol-12-myristate-acetate; 1 [micro]mol/l in 0.01% DMSO) successfully restored [P.sub.s] in ICAM-1 KO vessels to be not different from that of the WT controls. On the other hand, during TNF-[alpha]-induced inflammation, [P.sub.s] in WT mice was significantly increased (2-fold in venules and 2.5-fold in arterioles) in a Src-dependent and PKC-independent manner. The blockade of Src (PP2; 2 [micro]mol/l in 0.01% DMSO) but not PKC significantly reduced the TNF-[alpha]-dependent increase in [P.sub.s]. We conclude that ICAM-1 plays an essential role in the regulation of [P.sub.s] in microvessels and that there are two separate (constitutive and inducible) signaling pathways that regulate permeability under normal and inflamed conditions. adhesion molecules; microvascular exchange

podosomes (also termed invadopodia in cancer cells) are actin-rich adhesion structures with matrix degradation activity that develop in various cell types. Despite their significant physiological importance, the molecular mechanism of podosome formation is largely unknown. In this study, we investigated the molecular mechanisms of podosome formation. The expression of various phosphoinositide-binding domains revealed that the podosomes in Src-transformed NIH3T3 (NIH-src) cells are enriched with Ptdlns(3,4)P2, suggesting an important role of this phosphoinositide in podosome formation. Live-cell imaging analysis revealed that Src-expression stimulated podosome formation at focal adhesions of NIH3T3 cells after Ptdlns(3,4)P2 accumulation. The adaptor protein Tks5/FISH, which is essential for podosome formation, was found to form a complex with Grb2 at adhesion sites in an Src-dependent manner. Further, it was found that N-WASP bound all SH3 domains of Tks5/FISH, which facilitated circular podosome formation. These results indicate that augmentation of the N-WASP-Arp2/3 signal was accomplished on the platform of Tks5/FISH-Grb2 complex at focal adhesions, which is stabilized by Ptdlns(3,4)P2.

Membrane-associated guanylate kinase (MAGUK) proteins interact with several synaptogenesistriggering adhesion molecules. However, direct evidence for the involvement of MAGUK proteins in synapse formation is lacking. In this study, we investigate the function of calcium/calmodulin-dependent serine protein kinase (CASK), a MAGUK protein, in dendritic spine formation by RNA interference. Knockdown of CASK in cultured hippocampal neurons reduces spine density and shrinks dendritic spines. Our analysis of the time course of RNA interference and CASK overexpression experiments further suggests that CASK stabilizes or maintains spine morphology. Experiments using only the CASK PDZ domain or a mutant lacking the protein 4.1-binding site indicate an involvement of CASK in linking transmembrane adhesion molecules and the actin cytoskeleton. We also find that CASK is SUMOylated. Conjugation of small ubiquitin-like modifier 1 (SUMO1) to CASK reduces the interaction between CASK and protein 4.1. Overexpression of a CASK-SUMO1 fusion construct, which mimicks CASK SUMOylation, impairs spine formation. Our study suggests that CASK contributes to spinogenesis and that this is controlled by SUMOylation.

Rapid nerve impulse conduction in myelinated axons requires the concentration of voltage-gated sodium channels at nodes of Ranvier. Myelin-forming oligodendrocytes in the central nervous system (CNS) induce the clustering of sodium channels into nodal complexes flanked by paranodal axoglial junctions. However, the molecular mechanisms for nodal complex assembly in the CNS are unknown. Two isoforms of Neurofascin, neuronal Nfasc186 and glial Nfasc155, are components of the nodal and paranodal complexes, respectively. Neurofascin-null mice have disrupted nodal and paranodal complexes. We show that transgenic Nfasc186 can rescue the nodal complex when expressed in [Nfasc.sup.-/-] mice in the absence of the Nfasc155--Caspr--Contactin adhesion complex. Reconstitution of the axoglial adhesion complex by expressing transgenic Nfasc155 in oligodendrocytes also rescues the nodal complex independently of Nfasc186. Furthermore, the Nfasc155 adhesion complex has an additional function in promoting the migration of myelinating processes along CNS axons. We propose that glial and neuronal Neurofascins have distinct functions in the assembly of the CNS node of Ranvier.

Actin polymerizes near the leading edge of nerve growth cones, and actin filaments show retrograde movement in filopodia and lamellipodia. Linkage between actin filament retrograde flow and cell adhesion molecules (CAMs) in growth cones is thought to be one of the mechanisms for axon outgrowth and guidance. However, the molecular basis for this linkage remains elusive. Here, we show that shootin1 interacts with both actin filament retrograde flow and L1-CAM in axonal growth cones of cultured rat hippocampal neurons, thereby mediating the linkage between them. Impairing this linkage, either by shootin1 RNA interference or disturbing the interaction between shootin1 and actin filament flow, inhibited L1-dependent axon outgrowth, whereas enhancing the linkage by shootin1 overexpression promoted neurite outgrowth. These results strengthen the actin flow-CAM linkage model ('clutch' model) for axon outgrowth and suggest that shootin1 is a key molecule involved in this mechanism.

This study aimed to 1) assess whether substance P (SP) acts via neurokinin (NK)-I and NK-2 receptors to stimulate neurogenic inflammation (indicated by formation of ICAM-1 expression and oxidative stress) following oil smoke exposure (OSE) in rats; and 2) determine if pretreatment with antioxidants ameliorates the deleterious effects of OSE. Rats were pretreated with NK-1 receptor antagonist CP-96345, NK-2 receptor antagonist SR-48968, vitamin C, or catechins. OSE was for 30-120 min. Rats were killed 0-8 h later. Total lung resistance ([R.sub.L), airway smooth muscle activity (ASMA), lung ICAM-1 expression, neurogenic plasma extravasation (via India ink and Evans blue dye), bronchoalveolar lavage fluid SP concentrations, and reactive oxygen species formation [via lucigenin- and luminalamplified chemiluminescence (CL)] were assessed. Lung histology was performed. SP concentrations increased significantly in nonpretreated rats following OSE in a close-dependent manner. [R.sub.L] and total ASMA increased over time after OSE. Vitamin C and catechin pretreatments were associated with significantly reduced lucigenin CL 2 and 4 h after OSE. Pretreatment with catechins significantly reduced luminal CL counts 4 and 8 h after OSE. Evans blue levels were significantly reduced following 60 and 120 min of OSE in catechinand CP-96345-pretreated rats. ICAM-1 protein expression was significantly decreased in all pretreatment groups after OSE. Thickening of the alveolar capillary membrane, focal hemorrhaging, interstitial pneumonitis, and peribronchiolar inflammation were apparent in OSE lungs. These findings suggest that SP acts via the NK-1 receptor to provoke neurogenic inflammation, oxidative stress, and ICAM-1 expression after OSE in rats. reactive oxygen species; intracellular adhesion molecule-1: acute lung injury

Neuroligins (NLs) are postsynaptic cell-adhesion molecules that are implicated in humans in autism spectrum disorders because the genes encoding NL3 and NL4 are mutated in rare cases of familial autism. NLs are highly conserved evolutionarily, except that no NL4 was detected in the currently available mouse genome sequence assemblies. We now demonstrate that mice express a distant NL4 variant that rapidly evolved from other mammalian NL4 genes and that exhibits sequence variations even between different mouse strains. Despite its divergence, mouse NL4 binds neurexins and is transported into dendritic spines, suggesting that the core properties of NLs are retained in this divergent NL isoform. The selectively rapid evolution of NL4 in mice suggests that its function in the brain is under less stringent control than that of other NLs, shedding light on why its mutation in autism spectrum disorder patients is not lethal, but instead leads to a discrete developmental brain disorder. autism | cell-adhesion molecule | neurexin | synapse

The larval body wall muscles of Drosophila melanogaster arise by fusion of founder myoblasts (FMs) and fusion-competent myoblasts (FCMs). Sticks-and-Stones (SNS) is expressed on the surface of all FCMs and mediates adhesion with FMs and developing syncytia. Intracellular components essential for myoblast fusion are then recruited to these adhesive contacts. In the studies herein, a functional analysis of the SNS cytodomain using the GAL4/UAS system identified sequences that direct myoblast fusion, presumably through recruitment of these intracellular components. An extensive series of deletion and site-directed mutations were evaluated for their ability to rescue the myoblast fusion defects of sns mutant embryos. Deletion studies revealed redundant functional domains within SNS. Surprisingly, highly conserved consensus sites for binding post-synaptic density-95/discs large/zonula occludens-l-domain-containing (PDZ) proteins and serines with a high probability of phosphorylation play no significant role in myoblast fusion. Biochemical studies establish that the SNS cytodomain is phosphorylated at multiple tyrosines and their site-directed mutagenesis compromises the ability of the corresponding transgenes to rescue myoblast fusion. Similar mutagenesis revealed a requirement for conserved proline-rich regions. This complexity and redundancy of multiple critical sequences within the SNS cytodomain suggest that it functions through a complex array of interactions that likely includes both phosphotyrosine-binding and SH3-domain-containing proteins.

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between [[beta].sub.2]-integrin (lymphocyte function-associated antigen-1 and/or Mac-l) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of [[beta].sub.2]-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of [[beta].sub.2]-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions. heterotypic cell aggregation; adhesion molecule; leukocyte; tumor cell; reverse rate; binding affinity; probabilistic model; polymorpho-nuclear neutrophils; intercellular adhesion molecule-1

Correct targeting of proteins to axons and dendrites is crucial for neuronal function. We showed previously that axonal accumulation of the cell adhesion molecule L1/neuron-glia cell adhesion molecule (NgCAM) depends on endocytosis (Wisco, D., E.D. Anderson, M.C. Chang, C. Norden, T. Boiko, H. Folsch, and B. Winckler. 2003. J. Cell Biol. 162:1317-1328). Two endocytosis-dependent pathways to the axon have been proposed: transcytosis and selective retrieval/ retention. We show here that axonal accumulation of L1/NgCAM occurs via nondegradative somatodendritic endosomes and subsequent anterograde axonal transport, which is consistent with transcytosis. Additionally, we identify the neuronal-specific endosomal protein NEEP21 (neuron-enriched endosomal protein of 21 kD) as a regulator of L1/NgCAM sorting in somatodendritic endosomes. Down-regulation of NEEP21 leads to missorting of L1/NgCAM to the somatodendritic surface as well as to lysosomes. Importantly, the axonal accumulation of endogenous L1 in young neurons is also sensitive to NEEP21 depletion. We propose that small endosomal carriers derived from somatodendritic recycling endosomes can serve to redistribute a distinct set of membrane proteins from dendrites to axons.

The L1 cell adhesion molecule (L1CAM) participates in neuronal development. Mutations in the human L1 gene can cause the neurological disorder CRASH (corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraplegia, and hydrocephalus). This study presents genetic data that shows that L1-like adhesion gene 2 (LAD-2), a Caenorhabditis elegans L1CAM, functions in axon pathfinding. In the SDQL neuron, LAD-2 mediates dorsal axon guidance via the secreted MAB-20/Sema2 and PLX-2 plexin receptor, the functions of which have largely been characterized in epidermal morphogenesis. We use targeted misexpression experiments to provide in vivo evidence that MAB-20/Sema2 acts as a repellent to SDQL. Coimmunoprecipitation assays reveal that MAB-20 weakly interacts with PLX-2; this interaction is increased in the presence of LAD-2, which can interact independently with MAB-20 and PLX-2. These results suggest that LAD-2 functions as a MAB-20 coreceptor to secure MAB-20 coupling to PLX-2. In vertebrates, L1 binds neuropilinl, the obligate receptor to the secreted Sema3A. However, invertebrates lack neuropilins. LAD-2 may thus function in the semaphorin complex by combining the roles of neuropilins and L1 CAMs.

The pathogenesis of acute renal failure (ARF) occurring during the course of sepsis is incompletely understood. Intercellular adhesion molecule-1 (ICAM-1) is a key cell adhesion molecule upregulated by LPS, which binds to the integrins CD11a/CD18 and CD11b/CD18 present on the surface of leukocytes. We hypothesized that ICAM-1 facilitates renal injury in LPS-induced ARF. To test this, three groups of mice (n = 8 per group) were injected intraperitoneally with 6 mg/kg LPS: 1) normal C57BL/6 mice, 2) mice with a targeted deficiency of ICAM-1 ([ICAM-1.sup.-/-]), and 3) mice expressing very low levels of CD18 (CD18-def). [ICAM-1.sup.-/-] mice were significantly resistant to LPS-mediated ARF, as opposed to CD18-def mice, which developed severe ARF, as did wild-type controls (48 h blood urea nitrogen 143 [+ or -] 31.5, 70.8 [+ or -] 24.4, and 185 [+ or -] 16.6 mg/dl in wild-type, [ICAM-1.sup.-/-], and CD18-def mice, respectively, P < 0.05). At death, [ICAM-1.sup.-/-] mice had significantly less renal neutrophil infiltration than the other two groups, as well as less histological tubular injury. Depletion of neutrophils with mAb Gr-1 led to a profound exaggeration of tumor necrosis factor (TNF) release and high mortality, but neutrophil-depleted mice receiving 10-fold less LPS were protected against ARF despite TNF release similar to what is normally associated with LPS-induced ARF. LPS caused a significant increase in renal expression of chemokines; however, this increase was significantly exaggerated in CD18-def mice, which may account for their lack of protection. In conclusion, these data show that ICAM-1 plays a key role in LPS-induced ARF. sepsis; neutrophils; vascular cell adhesion molecule-1; chemokines

Modulation of adhesion molecule expression or function is regarded as a promising therapy for inflammatory conditions. This study evaluates the effects of an inhibitor of adhesion molecule expression (GI270384X) in two experimental models of colitis. Colitis of different severity was induced in C57BL/6J mice by administering 1, 2, or 3% dextran sulfate sodium (DSS). GI270384X (3, 10, or 25 mg x [kg.sup.-1] x [day.sup.-1]) was administered as pretreatment or started 3 days after colitis induction. In IL-10-deficient mice, the highest dose was given for 2 wk. The clinical course of colitis, pathological changes, serum inflammatory biomarkers, expression of adhesion molecules, and leukocyteendothelial cell interactions in colonic venules were measured in mice treated with vehicle or with active drug. In the most severe forms of colitis (2% and 3% DSS and IL-10-deficient mice), the magnitude of colonic inflammation was not modified by treatment with GI270384X. In a less severe form of colitis (1% DSS), GI270384X treatment dose dependently ameliorated the clinical signs of colitis, colonic pathological changes, and serum levels of biomarkers (IL-6 and serum amyloid A). Administration of 25 mg x [kg.sup.-1] x [day.sup.-1] GI270384X abrogated upregulation of ICAM-1 in the inflamed colon but had no effect on VCAM-1 or E-selectin expression. This was associated with a significant reduction in number of rolling and firmly adherent leukocytes in colonic venules. These results indicate that GI270384X is effective in the treatment of experimental colitis of moderate severity. Reduced adhesion molecule expression and leukocyte recruitment to the inflamed intestine contribute to this beneficial effect. endothelium; ICAM-1; leukocyte

Byline: Pelagia G. Tsoutsou (1,4), Konstantinos I. Gourgoulianis (1), Efthymia Petinaki (2), Maria Mpaka (1), Smaragda Efremidou (2), Antonios Maniatis (2), Pashalis Adam Molyvdas (3) Keywords: idiopathic pulmonary fibrosis; cell adhesion molecules; interstitial lung disease Abstract: In order to test the serum levels of ICAM-1, ICAM-2 and ICAM-3 in patients with idiopathic pulmonary fibrosis (IPF), twenty patients with IPF and eleven with secondary interstitial fibrosis (SIF), as well as forty healthy volunteers (HV) were studied. Serum intracellular adhesion molecules (ICAM) 1, 2 and 3 were assessed by ELISA. Functional respiratory tests, which included spirometry and lung diffusing capacity were simultaneously performed. Median values of serum ICAM-1 and ICAM-2 were higher in the patients' than in the healthy volunteers' (HV) group: IPF group: 946.60 ng/ml and 400.14 ng/ml SIF group: 901.58 ng/ml and 378.27 ng/ml HV group: 308.40 ng/ml and 217.55 ng/ml, respectively (p &lt 0.05). ICAM-3 serum levels were equal between the three groups. ICAM-2 negatively correlated to DLCO values. (p &lt 0.005). It can be concluded that ICAM 1 and 2 are elevated in the sera of patients with pulmonary fibrosis. ICAM-2 might be associated with a more impaired clinical status. Author Affiliation: (1) Pulmonary Department, University of Thessaly, Greece (2) Department of Microbiology, University of Thessaly, Greece (3) Physiology Department, University of Thessaly, Greece (4) Nikis 33, Syntagma Athens, 105 57, Greece Article History: Registration Date: 01/01/2004

Byline: Satoshi Gando (1), Takashi Kameue (1), Naoyuki Matsuda (1), Atsushi Sawamura (1), Mineji Hayakawa (1), Hirokatsu Kato (1) Keywords: acute lung injury (ALI); acute respiratory distress syndrome (ARDS); coagulation; inflammation; neutrophil; soluble adhesion molecules Abstract: To determine the existence of a close link between inflammation and coagulation in patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) and to examine their prognostic value in the development of ARDS and clinical outcome, we made a prospective cohort study. The study subjects consisted of 57 patients: 19 patients with ARDS and 38 patients with ALI as defined by a Lung Injury Score of aY=2.5 and 1.0 to less than 2.5, respectively. According to the outcome, the patients were subdivided into the survivors and the nonsurvivors. Ten normal healthy volunteers served as control subjects. Plasma levels of soluble L-, P-, and E-selectins, intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), thrombomodulin (sTM), and neutrophil elastase were measured within 24 h after the diagnosis of ALI or ARDS. The number of systemic inflammatory response syndrome (SIRS) criteria being met by the patients and the disseminated intravascular coagulation (DIC) scores were determined simultaneously. The number of SIRS criteria and the DIC scores of the patients with ALI or ARDS showed high values, and more than half of the patients were complicated by DIC. The levels of sL-selectin in both groups of the patients were significantly lower than those of the control subjects. All other soluble adhesion molecules, neutrophil elastase, and sTM in the patients with ALI and ARDS were markedly elevated than those in the control subjects. The levels sICAM-1, sVCAM-1, and sTM in the ARDS patients significantly increased compared with the ALI patients. The number of SIRS criteria and the DIC scores in the nonsurvivors showed higher values than those in the survivors. In addition, we found significant differences in the levels of soluble adhesion molecules, neutrophil elastase, and sTM between the survivors and the nonsurvivors. {In conclusion, we found a concurrent activation of both inflammation and coagulation in the patients with ALI or ARDS. The results also suggest that systemic activation of inflammation and coagulation associated with endothelial injury has prognostic value for the development of ARDS and poor outcome.} Author Affiliation: (1) Division of Acute and Critical Care Medicine, Department of Anesthesiology and Critical Care Medicine, Hokkaido, University Graduate School of Medicine, N15 W7, Kita-ku, Sapporo, Japan Article History: Registration Date: 20/12/2004

Byline: Lauree R. Thompson (1), Susan E. Keeney (1), Donna Tarrant (1), Elizabeth Rudloff (1), Dara V. Dallas (1), Frank C. Schmalstieg (1) Keywords: inflammation; endotoxin; E-selectin; P-selectin; soluble L-selectin Abstract: Sublethal doses of LPS result in increased tolerance to high concentrations of oxygen and this is associated with decreased pulmonary inflammation in a rat model. To investigate the mechanism of decreased neutrophil influx into the lung in this model, we measured levels of mRNA in the lung for the endothelial adhesion molecules, E-selectin and P-selectin. Immunostaining for E-selectin protein was also done in rat lungs, as well as measurement of soluble L-selectin in the blood. These levels were measured in the lungs of adult rats injected with 0.5 mg/kg LPS or placebo at 0 and 24 h and exposed to &gt 95% O.sub.2 for 60 h. Oxygen exposure resulted in significant increases in both E- and P-selectin mRNA and in E-selectin protein after 60 h. LPS resulted in an early rise in E-selectin protein followed by a decline to less than control (saline/O.sub.2) levels at 60 h. Messenger RNA for E-selectin followed a similar trend, although there were no differences at 60 h between LPS and control groups exposed to O.sub.2. P-selectin mRNA expression did not significantly differ between LPS and control O.sub.2 groups. Soluble L-selectin levels decreased by 6 h after LPS infusion and were significantly lower than saline/O.sub.2 controls through 24 h, suggesting binding to endothelium. In conclusion, the decrease in E-selectin expression on the surface of pulmonary endothelium after LPS could contribute to decreased inflammation in this model of oxygen toxicity. Soluble L-selectin may serve a further anti-inflammatory role after LPS infusion by binding to pulmonary endothelium. Author Affiliation: (1) Departments of Pediatrics and Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Texas Article History: Registration Date: 08/10/2004

Byline: K. A. Mohammed (1), N. Nasreen (1), J. Hardwick (1), R. D. Horn (1), K. L. Sanders (1), V. B. Antony (1) Keywords: Vascular endothelial growth factor; Mesothelial cells; Mycobacterium; N-cadherin; [beta]-catenin; Protein permeability Abstract: Patients with pulmonary tuberculosis develop pleural effusions with a high protein content. Pleural mesothelial adherens junctions promote mesothelial cell-cell adhesion and contribute to pleural integrity. In the present study we have investigated the effect of mycobacterium (BCG) on mesothelial cell adherens junction proteins and pleural permeability. BCG enhanced pleural mesothelial cell (PMC) release of vascular endothelial growth factor (VEGF), and decreased electrical resistance across the PMC monolayer. Neutralizing antibodies to VEGF significantly restored the drop in PMC electrical resistance caused by BCG. BCG infection down regulated [beta]-catenin (adherens junction protein) expression and caused increased permeability across confluent mesothelial monolayer. Our results suggest that in TB pleurisy, mycobacteria cause VEGF release from mesothelial cells and leads to protein exudation by altering mesothelial adherens junction proteins. Author Affiliation: (1) Division of Pulmonary and Critical Care Medicine, Department of Medicine, Veterans&apos; Affairs Medical Center, Indiana University School of Medicine, 1481 West 10th Street, 111-P, Indianapolis, IN 46202, USA Article History: Accepted Date: 10/02/2003

Byline: Karl-Gustav Kolbeck (1,2), Eliane Sardh (3), Hans Gyllenhammar (3,4), Lena Palmberg (5), Kjell Larsson K (5), Jan Palmblad (3) Keywords: Airway inflammation; swine dust; nitric oxide; adhesion molecules Abstract: Short-time exposure to swine dust causes an intense airways inflammation and symptoms of systemic inflammation in healthy volunteers. Here, we sought to study whether this response involved signs of endothelial cell activation. Peripheral blood cell counts and plasma levels of sE-selectin, sP-selectin, sICAM-1, interleukin-8, nitrite and nitrate were measured in blood samples from 17 healthy subjects before and after a 3-hr exposure to swine dust in a swine confinement building. Dust exposure induced a 3-fold increase of blood neutrophil p = 0.0009) and 1.5-fold increase of monocyte counts (p = 0.0047). IL-8 was detected in 15 individuals after exposure (p = 0.001). Endothelial cell markers such as sICAM and nitrate increased by 10 and 34% resp. (p = 0.011 and 0.017), whereas sE-selectin remained unchanged and sP-selectin was reduced by 15% (p = 0.031). Thus, short time exposure to swine dust induced a systemic inflammatory response with evidence of endothelial and inflammatory cell activation. Author Affiliation: (1) Division of Respiratory Medicine and Allergology at Department of Medicine, Huddinge University Hospital, Huddinge, Sweden (2) The Department of Respiratory Medicine and Allergology, Huddinge University Hospital, S-141 86, Stockholm, Sweden (3) Center for Inflammation and Hematology Research, Clinical Research Center, Novum, Sweden (4) Division of Hematology at Department of Medicine, Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden (5) National Institute for Working Life, Solna, Sweden Article History: Registration Date: 12/10/2004

Byline: Yuji Saito (1), Chizuko Nakagawa (1), Hiromi Uchida (1), Fumihiko Sasaki (1), Hiroki Sakakibara (1) Keywords: adhesion molecules; cAMP; flow cytometry Abstract: In this study we investigated the effect of adrenomedullin (AM) on fMLP-mediated activation of human neutrophils. AM partially, but significantly, suppressed fMLP-induced upregulation of CD11b expression. The inhibitory effects of AM upon fMLP-induced upregulation of CD11b expression were completely blocked by CGRP [8--37], a CGRP receptor antagonist. AM significantly increased cAMP content in neutrophils and SQ-22,536, an adenylate cyclase inhibitor, and KT-5720, a PKA inhibitor, significantly blocked the inhibitory effects of AM upon fMLP-induced upregulation of CD11b expression. This study indicates that binding of AM to the CGRP receptor suppresses fMLP-induced upregulation of CD11b expression of human neutrophils by increasing intracellular cAMP levels. AM may play an important role in the regulation of inflammatory processes, especially in the binding of neutrophils to vascular endothelial cells and subsequent neutrophil emigration evident in acute pulmonary inflammation. Author Affiliation: (1) Division of Respiratory Medicine and Allergy, Fujita Health University School of Medicine, 1--98, Dengakugakubo, Kutsukake, Toyoake-city, Aichi, 470--1192, Japan Article History: Registration Date: 17/10/2004

Byline: Helena Yusuf-Makagiansar (1), Irwan T. Makagiansar (1), Teruna J. Siahaan (1) Keywords: leukocyte infiltration; T-lymphocytes; adhesion molecules; apoptosis; LFA-1-peptide Abstract: Tissue inflammation is characterized by aggravated leukocyte infiltration into the sites of inflammation. The mechanism requires the interactions of leukocyte adhesion-molecules and their ligands in the inflamed tissues. In this study, we demonstrate that a cyclic peptide cLAB.L [cyclo1, 12-PenITDGEATDSGC], derived from the 'inserted' or I-domain of LFA-1 is able to inhibit the adherence of T-lymphocytes to the epithelial cell monolayers. This inhibition has been thought to involve the disruption of LFA-1/ICAM-1 interaction. The heterotypic adhesion of phorbol ester-activated Molt-3 cells and IFN-I3-induced Caco-2 monolayers was inhibited upon treatment of the monolayers with monoclonal antibodies (MAbs) to adhesion molecules or with cLAB.L peptide. The adhesion can be inhibited by MAbs to ICAM-1, ICAM-2, and VCAM-1, and cLAB.L peptide in a concentration-dependent manner. However, none of the individual uses of these molecules led to a total inhibition. The inhibitory activity of cLAB.L is greatly reduced by low temperature and the absence of cell activation. Treatment of cLAB.L peptide may trigger an early event of apoptosis on activated but not on non-activated Molt-3 cells no indication of peptide-induced apoptosis was found on Caco-2 cells. Taken together, data from this work suggest that cLAB.L may have applications to direct cell-targeted delivery during tissue inflammation. Author Affiliation: (1) Department of Pharmaceutical Chemistry, Simons Research Laboratories, The University of Kansas, 2095 Constant Avenue, Lawrence, Kansas, 66047 Article History: Registration Date: 17/10/2004

Byline: Giuseppe Montalto (1), Lydia Giannitrapani (1), Maurizio Soresi (1), Lucrezia Virruso (2), Daniela di Martino (1), Roberto Gambino (2), Antonio Carroccio (1), Melchiorre Cervello (2) Keywords: chronic liver disease; E-selectin; adhesion molecule; treatment; [alpha]-interferon Abstract: E-selectin, an adhesion molecule of the selectin family, is involved in leukocyte adhesion to the endothelium and in the cellular immunological reactions. Expression of this molecule, in fact, is physiologically absent, but it becomes evident on sinusoidal lining cells during inflammatory liver disease. The aim of this study was to evaluate the behavior of E-selectin in chronic hepatitis C (CH-C) patients with persistently normal transaminase in comparison to patients with CH-C and elevated transaminase, and its changes during alpha-interferon therapy. Immunohistochemical localization of E-selectin was also performed on liver tissue specimens of both groups. Fifty-eight subjects were divided into 3 groups: group A included 18 patients with CH-C and persistently normal transaminase group B 20 patients with CH-C and persistently elevated transaminase levels and group C included 20 healthy subjects, representing the control group. The first two groups were treated with r-IFN [alpha] at a dose of 6 MU 3 times a week for 3 months and followed-up with 3 MU 3 times a week for another 3 months. Serum baseline values of E-selectin in groups A and B were significantly higher than those in group C (P &lt 0.04), but there was no difference between groups A and B. Furthermore, there was a trend toward higher E-selectin values as histological severity increased (r = 0.69 P &lt 0.0001). Post-treatment E-selectin serum values showed a moderate decrease in both groups, but only among responder patients while E-selectin levels were unchanged in non responders. Immunohistochemical localization showed no staining for E-selectin in normal liver specimens, while there was a quite similar staining for E-selectin in the two groups of patients. In conclusion, this study shows that serum E-selectin levels in patients with CH-C and persistently normal transaminase are higher than in controls and they are associated with severity of liver disease. Liver of these patients express E-selectin molecules, suggesting an activation of the immune system almost identical to that of patients with CH-C and elevated transaminase. In both groups only responder patients showed a moderate decrease below baseline serum values. Author Affiliation: (1) Istituto di Medicina Interna e Geriatria, Universita di Palermo, via del Vespro 141, 90127 Cattedra di Medicina Interna, Palermo, Italy (2) Istituto di Biologia dello Sviluppo, Consiglio Nazionale delle Ricerche (CNR), Palermo, Italy Article History: Registration Date: 17/10/2004

Byline: Masako Ohnishi (1), Noriaki Imanishi (1) Abstract: The role of P-selectin in the accumulation of neutrophils at acute dermal inflammatory sites induced by chemoattractants, LTB4 and IL-8 was investigated in the mouse. A mouse P-selectin-human IgG chimera bound to mouse neutrophils in vitro in a calcium-dependent manner, as detected by flow cytometry. A rat monoclonal antibody (mAb) against mouse P-selectin, RB40.34 abolished P-selectin-IgG chimera binding to mouse neutrophils, but a control antibody did not. Intradermal injection of LTB4 at a dose of 100 ng/site caused neutrophil accumulation to increase by 3-4 fold, as detected by measuring myeloperoxidase activity. Neutrophil extravasation to perivascular tissue was detected by histochemical observation. The intravenous injection of RB40.34 or the specific LTB4 antagonist, SM-15178, at doses at 5 mg/kg attenuated the accumulation of neutrophils by 55.6% and 70.3%, respectively, but a control antibody showed no effect. Similarly, intradermal administration of IL-8 at a dose of 5 ug/site induced significant neutrophil migration into the interstitial tissue of the skin, as followed by measuring myeloperoxidase activity and histopathologic analysis. The intravenous injection of RB40.34 at a dose of 5 mg/kg reduced the neutrophil accumulation by 59.2% in contrast, a control antibody showed no effect. To our knowledge, this is the first direct demonstration that P-selectin plays a substantial role in LTB4and IL-8-induced neutrophil accumulation in mouse skin. Author Affiliation: (1) Research Center, Sumitomo Pharmaceuticals, 1-98, Kasugadenaka 3-chome, Konohana-ku, Osaka, 554--0022, Japan Article History: Registration Date: 05/10/2004

Byline: Andrea K. Hubbard (1), Charles Giardina (2) Abstract: In a mouse model of silica (SI) induced lung injury, SI exposure increases expression of intercellular adhesion molecule-1 (ICAM-1) on lung (alveolar/interstitial) macrophages and alveolar type II epithelial cells. To investigate the regulation of SI induced ICAM-1 expression on mouse macrophages, freshly isolated macrophages (alveolar, peritoneal) and macrophage cell lines (MH-S, RAW 264.7) were evaluated for ICAM-1 expression elicited by the particle silica ([alpha] quartz 20 ug/ml 6 ug/cm.sup.2) or the inflammatory cytokine, TNF[alpha] (20 ng/ml). TNF[alpha] significantly increased ICAM-1 expression in all cell types whereas SI elicited an increase in peritoneal macrophages (PM) and the cell line, MH-S. This pattern of increased expression was confirmed by immunocytochemistry. To investigate the regulation of ICAM-1 expression, PM were incubated with SI, TNF[alpha] or media concomitantly with anti-TNF[alpha] antibody, the antioxidant, NAC, or the iNOS synthase inhibitor, L-NAME. Both anti-TNF[alpha] and NAC, but not L-NAME, inhibited elicited (TNF[alpha], SI) as well as constitutive (media) ICAM-1 expression. These data demonstrate that both inflammatory cytokines and inorganic particles can increase ICAM-1 expression on mouse macrophages and that this expression is mediated, in part, by TNF[alpha] and reactive oxygen species. Author Affiliation: (1) Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut, 06269 (2) Department of Molecular and Cellular Biology, University of Connecticut, Storrs, Connecticut, 06269 Article History: Registration Date: 09/10/2004