Creating high-quality radiology reports in foreign languages through multilingual structured reporting.

OBJECTIVES: Globalization and migration are increasing the demand for reports in different languages. We aimed to examine if structured reports created by non-German-speaking radiologists with multilingual templates show significant differences in quality to structured reports and free-text reports by German native speakers. METHODS: We used structured templates that allow radiologists to report in their mother tongue and then switch the report language to German or English automatically using proprietary software. German- and English-speaking radiology residents created structured reports in both German and English with these templates. Reports for three different exam types were created (intensive care chest x-ray, shoulder x-ray specifically for degenerative processes, and CT pulmonary angiogram for pulmonary embolism). The report quality of automatically translated German structured reports by English-speaking radiologists and German structured reports by German radiologists was then evaluated by German clinicians with a standardized questionnaire. The questionnaire was designed to assess attributes including content, comprehensibility, clinical consequences, and overall quality. RESULTS: Structured reports by English-speaking radiologists that were automatically translated into German and German structured reports by German radiologists both received very high or high overall quality ratings in the majority of cases, showing no significant differences in quality. Likewise, no significant differences were observed between the two report types regarding comprehensibility and clinical consequences. Structured reports by German radiologists received significantly better ratings for overall quality and comprehensibility compared to free-text reports by German radiologists. CONCLUSIONS: Multilingual structured reporting templates may serve as a feasible tool for creating high-quality radiology reports in foreign languages. KEY POINTS: • Multilingualism in structured reporting templates can be a useful tool for creating high-quality radiology reports in foreign languages. • German reports created with multilingual structured reporting templates by English-speaking radiologists and German structured reports by German radiologists exhibit no significant differences in overall report quality. • Multilingual structured reporting templates can help radiologists overcome communication barriers and facilitate teleradiology.

Aeshnid dragonfly larvae as bioindicators of methylmercury contamination in aquatic systems impacted by elevated sulfate loading.

Methylmercury (MeHg) levels in dragonfly larvae and water were measured over two years in aquatic systems impacted to varying degrees by sulfate releases related to iron mining activity. This study examined the impact of elevated sulfate loads on MeHg concentrations and tested the use of MeHg in dragonfly larvae as an indicator of MeHg levels in a range of aquatic systems including 16 river/stream sites and two lakes. MeHg concentrations in aeshnid dragonfly larvae were positively correlated (R(2) = 0.46, p < 0.01) to peak MeHg concentrations in the dissolved phase for the combined years of 2012 and 2013. This relation was strong in 2012 (R(2) = 0.85, p < 0.01), but showed no correlation in 2013 (R(2) = 0.02, p > 0.05). MeHg in dragonfly larvae were not elevated at the highest sulfate sites, but rather the reverse was generally observed. Record rainfall events in 2012 and above average rainfall in 2013 likely delivered the majority of Hg and MeHg to these systems via interflow and activated groundwater flow through reduced sediments. As a result, the impacts of elevated sulfate releases due to mining activities were not apparent in these systems where little of the sulfate is reduced. Lower bioaccumulation factors for MeHg in aeshnid dragonfly larvae were observed with increasing dissolved organic carbon (DOC) concentrations. This finding is consistent with previous studies showing that MeHg in high DOC systems is less bioavailable; an equilibrium model shows that more MeHg being associated with DOC rather than algae at the base of the food chain readily explains the lower bioaccumulation factors.

[Fax Survey to Elucidate the Information Needs of General Practitioners in Lower Saxony Regarding the Topic of Medical Implants]. / Faxumfrage zum Informationsbedarf niedersächsischer Hausärzte zum Thema "Medizinische Implantate".

Background: Medial implants help a multitude of patients to gain more health, mobility and thus, quality of life. In collaboration with a still growing expectation of life especially, i.e., within Western industrial countries, this has led to an increasing use of implants over the last years. However, although biomechanical characteristics of modern implant materials have improved considerably, one big challenge still exists - the implant-associated infection. Early diagnostic and therapeutic interventions could clearly mitigate this issue, but are general practitioners sufficiently informed regarding this topic? Material and Methods: In March 2013 and in close cooperation with the Lower Saxony association of general practitioners, we initiated a survey to elucidate the information demands of general practitioners regarding the topic of medical implants. A total of 939 members of the association were contacted via fax and 101 (10.8 %) responded. Based on the obtained data, we then evaluated which topics are most interesting for this group of medical professionals. Results: The survey clearly indicates that general practitioners request more general implant-related data, e.g., type and specification of an implant as well as its location within the individual patient and contact addresses of the implanting hospital, but also want more specific information regarding diagnostic and therapeutic strategies in the case of implant-associated complications. Conclusion: The present article reports in detail on the conducted fax survey and shows some initial strategies as to how the identified challenges might be faced.

Primary haemostasis: newer insights.

At the same time as biophysical and omics approaches are drilling deeper into the molecular details of platelets and other blood cells, as well as their receptors and mechanisms of regulation, there is also an increasing awareness of the functional overlap between human vascular systems. Together, these studies are redefining the intricate networks linking haemostasis and thrombosis with inflammation, infectious disease, cancer/metastasis and other vascular pathophysiology. The focus of this state-of-the-art review is some of the newer advances relevant to primary haemostasis. Of particular interest, platelet-specific primary adhesion-signalling receptors and associated activation pathways control platelet function in flowing blood and provide molecular links to other systems. Platelet glycoprotein (GP)Ibα of the GPIb-IX-V complex and GPVI not only initiate platelet aggregation and thrombus formation by primary interactions with von Willebrand factor and collagen, respectively, but are also involved in coagulation, leucocyte engagement, bacterial or viral interactions, and are relevant as potential risk markers in a range of human diseases. Understanding these systems in unprecedented detail promises significant advances in evaluation of individual risk, in new diagnostic or therapeutic possibilities and in monitoring the response to drugs or other treatment.

Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location.

Particulate air pollution is widespread, yet we have little understanding of the long-term health implications associated with exposure. We investigated DNA damage, mutation, and methylation in gametes of male mice exposed to particulate air pollution in an industrial/urban environment. C57BL/CBA mice were exposed in situ to ambient air near two integrated steel mills and a major highway, alongside control mice breathing high-efficiency air particulate (HEPA) filtered ambient air. PCR analysis of an expanded simple tandem repeat (ESTR) locus revealed a 1.6-fold increase in sperm mutation frequency in mice exposed to ambient air for 10 wks, followed by a 6-wk break, compared with HEPA-filtered air, indicating that mutations were induced in spermatogonial stem cells. DNA collected after 3 or 10 wks of exposure did not exhibit increased mutation frequency. Bulky DNA adducts were below the detection threshold in testes samples, suggesting that DNA reactive chemicals do not reach the germ line and cause ESTR mutation. In contrast, DNA strand breaks were elevated at 3 and 10 wks, possibly resulting from oxidative stress arising from exposure to particles and associated airborne pollutants. Sperm DNA was hypermethylated in mice breathing ambient relative to HEPA-filtered air and this change persisted following removal from the environmental exposure. Increased germ-line DNA mutation frequencies may cause population-level changes in genetic composition and disease. Changes in methylation can have widespread repercussions for chromatin structure, gene expression and genome stability. Potential health effects warrant extensive further investigation.

Basal Ganglia versus Peripheral Infarcts: Predictive Value of Early Fiber Alterations.

BACKGROUND AND PURPOSE: Impairment of fiber integrity of the corticospinal tract in the subacute and chronic phases after ischemic stroke has been linked to poor motor outcome. The aim of the study was an assessment of fiber integrity in the acute poststroke phase and an evaluation of its association with the clinical course dependent on the infarction pattern (subtypes: peripheral versus basal ganglia infarction). MATERIALS AND METHODS: All patients who underwent mechanical recanalization of a large-vessel occlusion in the anterior circulation and postinterventional DTI were included (n = 165). The fractional anisotropy index of the patient-specific corticospinal tract within the posterior limb of the internal capsule was correlated to clinical parameters (NIHSS scores/mRS at 90 days), and the interaction of stroke subtype (peripheral infarcts versus basal ganglia infarction) was tested in a moderation analysis. RESULTS: The fractional anisotropy index was reduced in the acute poststroke phase with a correlation to clinical presentation, especially in case of peripheral infarcts (eg, with the NIHSS motor subscore: r = -0.4, P < .001). This correlation was absent for basal ganglia infarction (r = -0.008, P > .05). There was a significant association between the fractional anisotropy index and clinical outcome (mRS after 90 days, P < .01), which is moderated by stroke subtype with significant effects only for peripheral infarcts. CONCLUSIONS: Corticospinal tract abnormalities can be observed in the early stage after mechanical recanalization and have prognostic capacity. This finding increases the clinical value of early DTI imaging parameters. Because the effects observed were limited to peripheral infarcts, further and longitudinal evaluation of fiber integrities within basal ganglia infarction is required.

A short history of thrombectomy - Procedure and success analysis of different endovascular stroke treatment techniques.

BACKGROUND: The historical development of interventional stroke treatment shows a wide variation of different techniques and materials used. Thus, the question of the present work is whether the technical and procedural differences of thrombectomy techniques lead to different technical and clinical results. METHODS AND RESULTS: Analysis of a mixed retrospective/prospective database of all endovascular treated patients with an occlusion of the Carotid-T or M1 segment of the MCA at a single comprehensive stroke center since 2008. Patients were classified regarding the technical approach used. Six hundred sixty-eight patients were available for the final analysis. Reperfusion rates ranged between 56% and 100% depending on the technical approach. The use of balloon guide catheters and most recently the establishment of combination techniques using balloon guide catheters, aspiration catheters and stent retrievers have shown a further significant increase in the rates of successful recanalization, full recanalization and first-pass recanalization. Additionally, the technical development of interventional techniques has led to a subsequent drop in complications, embolization into previously unaffected territories in particular. CONCLUSION: Technical success of MT has improved substantially over the past decade owing to improved materials and procedural innovations. Combination techniques including flow modulation have emerged to be the most effective approach and should be considered as a standard of care.Level of evidence: Level 3, retrospective study.

Thrombus Histology of Basilar Artery Occlusions : Are There Differences to the Anterior Circulation?

BACKGROUND: For patients with acute vessel occlusions of the anterior circulation histopathology of retrieved cerebral thrombi has been reported to be associated to stroke etiology. Due to the relatively small incidence of posterior circulation stroke, exclusive histopathologic analyses are missing for this subgroup. The aim of the study was to investigate thrombus histology for patients with basilar artery occlusions and uncover differences to anterior circulation clots with respect to underlying etiology. METHODS: A total of 59 basilar thrombi were collected during intracranial mechanical recanalization and quantitatively analyzed in terms of their relative fractions of the main constituents, e.g. fibrin/platelets (F/P), red (RBC) and white blood cells (WBC). Data were compared to histopathological analyses of 122 thrombi of the anterior circulation with respect to underlying pathogenesis. RESULTS: The composition of basilar thrombi differed significantly to thrombi of the anterior circulation with an overall higher RBC amount (median fraction in % (interquartile range):0.48 (0.37-0.69) vs. 0.37 (0.28-0.50), p < 0.001) and lower F/P count (0.45 (0.21-0.58) vs. 0.57 (0.44-0.66), p < 0.001). Basilar thrombi composition did not differ between the different etiological stroke subgroups. CONCLUSION: The results depict a differing thrombus composition of basilar thrombi in comparison to anterior circulation clots with an overall higher amount of RBC. This may reflect different pathophysiologic processes between anterior and posterior circulation thrombogenesis, e.g. a larger proportion of appositional thrombus growth in the posterior circulation.

P-selectin and platelet-activating factor mediate initial endotoxin-induced neutropenia.

Polymorphonuclear neutrophil (PMN) accumulation within damaged tissues, a hallmark of acute inflammation, is dependent upon initial adhesion to endothelial cells. In vitro studies suggest that P-selectin and platelet activating factor (PAF) are key molecules in this process by promoting the initial adhesion of PMN to endothelial cells. We report in vivo studies in which intravenous administration of lipopolysaccharide (LPS) to anesthetized rats caused a very rapid onset (< 5 min) of neutropenia, in association with induction of surface expression of P-selectin on microvascular endothelial cells in kidney, liver and lung; analogous induction of P-selectin expression by cultured endothelial cells was observed in response to LPS stimulation in vitro. In addition, treatment with an antibody (Ab) to P-selectin (or use of a PAF antagonist) blocked development of neutropenia in vivo for at least 15 min post-LPS injection, and Ab treatment was shown to block PMN accumulation in tissues. These studies document roles for P-selectin and PAF in the early adhesion of PMN to endothelial cells in vivo.

Platelet glycoprotein ibalpha is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18).

The firm adhesion and transplatelet migration of leukocytes on vascular thrombus are both dependent on the interaction of the leukocyte integrin, Mac-1, and a heretofore unknown platelet counterreceptor. Here, we identify the platelet counterreceptor as glycoprotein (GP) Ibalpha, a component of the GP Ib-IX-V complex, the platelet von Willebrand factor (vWf) receptor. THP-1 monocytic cells and transfected cells that express Mac-1 adhered to GP Ibalpha-coated wells. Inhibition studies with monoclonal antibodies or receptor ligands showed that the interaction involves the Mac-1 I domain (homologous to the vWf A1 domain), and the GP Ibalpha leucine-rich repeat and COOH-terminal flanking regions. The specificity of the interaction was confirmed by the finding that neutrophils from wild-type mice, but not from Mac-1-deficient mice, bound to purified GP Ibalpha and to adherent platelets, the latter adhesion being inhibited by pretreatment of the platelets with mocarhagin, a protease that specifically cleaves GP Ibalpha. Finally, immobilized GP Ibalpha supported the rolling and firm adhesion of THP-1 cells under conditions of flow. These observations provide a molecular target for disrupting leukocyte-platelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis.

Characterization of GMP-140 (P-selectin) as a circulating plasma protein.

GMP-140 is a 140-kD granule membrane protein, found in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells, that is surface expressed on cell activation and mediates neutrophil attachment. Cloning data for GMP-140 from an endothelial library predict a soluble form of the protein, the transcription message for which is also found in platelets. In this study, we report the detection by enzyme-linked immunosorbent assay of soluble GMP-140 in plasma centrifuged for 3 h at 100,000 g (to remove platelet microparticles) and confirm its identity by purification from plasma. Plasma concentrations were found to be 0.251 +/- 0.043 micrograms/ml (means +/- SD, n = 10) in normal male controls and 0.175 +/- 0.063 micrograms/ml (means +/- SD, n = 10) in normal female controls. The purified protein had an identical molecular mass (nonreduced) to platelet membrane GMP-140 (approximately 3 kD lower, reduced) and was immunoblotted by polyclonal anti-GMP-140, and the anti-GMP-140 monoclonal antibodies AK4 and AK6. Analytical gel filtration studies indicated that the plasma GMP-140 eluted as a monomer whereas detergent-free, platelet membrane GMP-140 eluted as a tetramer consistent with plasma GMP-140 lacking a transmembrane domain. Purified plasma GMP-140 bound to the same neutrophil receptor as the membrane-bound form, and when immobilized on plastic, bound neutrophils equivalently to immobilized platelet membrane GMP-140. Since it has been shown that fluid-phase GMP-140 is antiinflammatory and downregulates CD18-dependent neutrophil adhesion and respiratory burst, its presence in plasma may be of major importance in preventing the inadvertent activation of neutrophils in the circulation.

The glycoprotein Ib-IX-V complex is a platelet counterreceptor for P-selectin.

We have identified platelet glycoprotein (GP) Ibalpha as a counterreceptor for P-selectin. GP Ibalpha is a component of the GP Ib-IX-V complex, which mediates platelet adhesion to subendothelium at sites of injury. Cells expressing P-selectin adhered to immobilized GP Ibalpha, and GP Ibalpha-expressing cells adhered to and rolled on P-selectin and on histamine-stimulated endothelium in a P-selectin-dependent manner. In like manner, platelets rolled on activated endothelium, a phenomenon inhibited by antibodies to both P-selectin and GP Ibalpha. Unlike the P-selectin interaction with its leukocyte ligand, PSGL-1 (P-selectin glycoprotein ligand 1), the interaction with GP Ibalpha required neither calcium nor carbohydrate core-2 branching or alpha(1,3)-fucosylation. The interaction was inhibited by sulfated proteoglycans and by antibodies against GP Ibalpha, including one directed at a tyrosine-sulfated region of the polypeptide. Thus, the GP Ib-IX-V complex mediates platelet attachment to both subendothelium and activated endothelium.

Na+-dependent gate dynamics and electrostatic attraction ensure substrate coupling in glutamate transporters.

Excitatory amino acid transporters (EAATs) harness [Na+], [K+], and [H+] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na+ ions, [Na+] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na+:substrate coupling in the EAAT homolog GltPh A lipidic cubic phase x-ray crystal structure of wild-type, Na+-only bound GltPh at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na+ ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na+-stabilized gate opening and electrostatic coupling of aspartate to Na+ binding provides a constant Na+:substrate transport stoichiometry over a broad range of neurotransmitter concentrations.

Microstructural Integrity of Salvaged Penumbra after Mechanical Thrombectomy.

BACKGROUND AND PURPOSE: There are sparse data on the microstructural integrity of salvaged penumbral tissue after mechanical thrombectomy of large-vessel occlusions. The aim of the study was to analyze possible microstructural alteration in the penumbra and their association with clinical symptoms as well as angiographic reperfusion success in patients undergoing mechanical thrombectomy. MATERIALS AND METHODS: All patients who underwent mechanical thrombectomy for large-vessel occlusions in the anterior circulation and who received an admission CT perfusion together with postinterventional DTIs were included (n = 65). Angiographic reperfusion success by means of modified Thrombolysis in Cerebral Infarction (mTICI) scale and clinical outcome were recorded. Microstructural integrity was assessed by DTI evaluating the mean diffusivity index within the salvaged gray matter of the former penumbra. RESULTS: The mean diffusivity index was higher in completely recanalized patients (mTICI 3: -0.001 ± 0.034 versus mTICI <3: -0.030 ± 0.055, P = .03). There was a positive correlation between the mean diffusivity index and NIHSS score improvement (r = 0.49, P = .003) and the mean diffusivity index was associated with midterm functional outcome (r = -0.37, P = .04) after adjustment for confounders. In mediation analysis, the mean diffusivity index and infarction growth mediated the association between reperfusion success and clinical outcomes. CONCLUSIONS: The macroscopic salvaged penumbra included areas of microstructural integrity changes, most likely related to the initial hypoperfusion. These abnormalities were found early after mechanical thrombectomy, were dependent on angiographic results, and correlated with the clinical outcome. When confirmed, these findings prompt the evaluation of therapies for protection of the penumbral tissue integrity.

Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model.

We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.

Regulation of in vitro capillary tube formation by anti-integrin antibodies.

Human endothelial cells are induced to form an anastomosing network of capillary tubes on a gel of collagen I in the presence of PMA. We show here that the addition of mAbs, AK7, or RMAC11 directed to the alpha chain of the major collagen receptor on endothelial cells, the integrin alpha 2 beta 1, enhance the number, length, and width of capillary tubes formed by endothelial cells derived from umbilical vein or neonatal foreskins. The anti-alpha 2 beta 1 antibodies maintained the endothelial cells in a rounded morphology and inhibited both their attachment to and proliferation on collagen but not on fibronectin, laminin, or gelatin matrices. Furthermore, RMAC11 promoted tube formation in collagen gels of increased density which in the absence of RMAC11 did not allow tube formation. Neither RMAC11 or AK7 enhanced capillary formation in the absence of PMA. Lumen structure and size were also altered by antibody RMAC11. In the absence of antibody the majority of lumina were formed intracellularly from single cells, but in the presence of RMAC11, multiple cells were involved and the lumen size was correspondingly increased. Endothelial cells were also induced to undergo capillary formation in fibrin gels after PMA stimulation. The addition of anti-alpha v beta 3 antibodies promoted tube formation in fibrin gels and inhibited EC adhesion to and proliferation on a fibrinogen matrix. The enhancement of capillary formation by the anti-integrin antibodies was matrix specific; that is, anti-alpha v beta 3 antibodies only enhanced tube formation on fibrin gels and not on collagen gels while anti-alpha v beta 1 antibodies only enhanced tubes on collagen and not on fibrin gels. Thus we postulate that changes in the adhesive nature of endothelial cells for their extracellular matrix can profoundly effect their function. Anti-integrin antibodies which inhibit cell-matrix interactions convert endothelial cells from a proliferative phenotype towards differentiation which results in enhanced capillary tube formation.

Identification of a membrane skeleton in platelets.

Platelets have previously been shown to contain actin filaments that are linked, through actin-binding protein, to the glycoprotein (GP) Ib-IX complex, GP Ia, GP IIa, and an unidentified GP of Mr 250,000 on the plasma membrane. The objective of the present study was to use a morphological approach to examine the distribution of these membrane-bound filaments within platelets. Preliminary experiments showed that the Triton X-100 lysis buffers used previously to solubilize platelets completely disrupt the three-dimensional organization of the cytoskeletons. Conditions were established that minimized these postlysis changes. The cytoskeletons remained as platelet-shaped structures. These structures consisted of a network of long actin filaments and a more amorphous layer that outlined the periphery. When Ca2+ was present, the long actin filaments were lost but the amorphous layer at the periphery remained; conditions were established in which this amorphous layer retained the outline of the platelet from which it originated. Immunocytochemical experiments showed that the GP Ib-IX complex and actin-binding protein were associated with the amorphous layer. Analysis of the amorphous material on SDS-polyacrylamide gels showed that it contained actin, actin-binding protein, and all actin-bound GP Ib-IX. Although actin filaments could not be visualized in thin section, the actin presumably was in a filamentous form because it was solubilized by DNase I and bound phalloidin. These studies show that platelets contain a membrane skeleton and suggest that it is distinct from the network of cytoplasmic actin filaments. This membrane skeleton exists as a submembranous lining that, by analogy to the erythrocyte membrane skeleton, may stabilize the plasma membrane and contribute to determining its shape.

Novel cytokine-independent induction of endothelial adhesion molecules regulated by platelet/endothelial cell adhesion molecule (CD31).

Tumor necrosis factor-alpha, interleukin-1, and endotoxin stimulate the expression of vascular endothelial cell (EC) adhesion molecules. Here we describe a novel pathway of adhesion molecule induction that is independent of exogenous factors, but which is dependent on integrin signaling and cell-cell interactions. Cells plated onto gelatin, fibronectin, collagen or fibrinogen, or anti-integrin antibodies, expressed increased amounts of E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. In contrast, ECs failed to express E-selectin when plated on poly-L-lysine or when plated on fibrinogen in the presence of attachment-inhibiting, cyclic Arg-Gly-Asp peptides. The duration and magnitude of adhesion molecule expression was dependent on EC density. Induction of E-selectin on ECs plated at confluent density was transient and returned to basal levels by 15 h after plating when only 7 +/- 2% (n = 5) of cells were positive. In contrast, cells plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 +/- 4% (n = 5) positive for E-selectin expression 15 h after plating, and significant expression still evident 72 h after plating. The confluency-dependent inhibition of expression of E-selectin was at least partly mediated through the cell junctional protein, platelet/endothelial cell adhesion molecule-1 (PECAM-1). Antibodies against PECAM-1, but not against VE-cadherin, increased E-selectin expression on confluent ECs. Co- culture of subconfluent ECs with PECAM-1- coated beads or with L cells transfected with full-length PECAM-1 or with a cytoplasmic truncation PECAM-1 mutant, inhibited E-selectin expression. In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant failed to regulate E-selectin expression. In an in vitro model of wounding the wound front displayed an increase in the number of E-selectin-expressing cells, and also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer. Thus we propose that the EC junction, and in particular, the junctional molecule PECAM-1, is a powerful regulator of endothelial adhesiveness.

Activation-dependent changes in human platelet PECAM-1: phosphorylation, cytoskeletal association, and surface membrane redistribution.

PECAM-1 is a recently described member of the immunoglobulin gene (Ig) superfamily that is expressed on the surface on platelets, several leukocyte subsets, and at the endothelial cell intracellular junction. Recent studies have shown that the extracellular domain of PECAM-1, which is comprised of 6 Ig-like homology units, participates in mediating cell-cell adhesion, plays a role in initiating endothelial cell contact, and may later serve to stabilize the endothelial cell monolayer. PECAM-1 also has a relatively large 108 amino acid cytoplasmic domain, with potential sites for phosphorylation, lipid modification, and other posttranslational events that could potentially modulate its adhesive function or regulate its subcellular distribution. Virtually nothing is known about the contribution of the intracellular region of the PECAM-1 molecule to either of these cellular processes. Using human platelets as a model, we now demonstrate that PECAM-1 becomes highly phosphorylated in response to cellular activation, and coincident with phosphorylation associates with the cytoskeleton of activated, but not resting, platelets. The engagement of PECAM-1 with the platelet cytoskeleton enables it to move large distances within the plane of the membrane of fully-spread, adherent platelets. This redistribution may similarly account for the ability of PECAM-1 to localize to the intracellular borders of endothelial cells once cell-cell contact has been achieved.

The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin.

SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline-rich domain. To identify proteins that bind to the SHIP-2 proline-rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.