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Traumatic brain injury (TBI) remains a major cause of morbidity and death among the pediatric population. Timely diagnosis, however, remains a complex task because of the lack of standardized methods that permit its accurate identification. The aim of this study was to determine whether serum levels of brain injury biomarkers can be used as a diagnostic and prognostic tool in this pathology. This prospective, observational study collected and analyzed the serum concentration of neuronal injury biomarkers at enrollment, 24h and 48h post-injury, in 34 children ages 0-18 with pTBI and 19 healthy controls (HC). Biomarkers included glial fibrillary acidic protein (GFAP), neurofilament protein L (NfL), ubiquitin-C-terminal hydrolase (UCH-L1), S-100B, tau and tau phosphorylated at threonine 181 (p-tau181). Subjects were stratified by admission Glasgow Coma Scale score into two categories: a combined mild/moderate (GCS 9-15) and severe (GCS 3-8). Glasgow Outcome Scale-Extended (GOS-E) Peds was dichotomized into favorable (≤4) and unfavorable (≥5) and outcomes. Data were analyzed utilizing Prism 9 and R statistical software. The findings were as follows: 15 patients were stratified as severe TBI and 19 as mild/moderate per GCS. All biomarkers measured at enrollment were elevated compared with HC. Serum levels for all biomarkers were significantly higher in the severe TBI group compared with HC at 0, 24, and 48h. The GFAP, tau S100B, and p-tau181 had the ability to differentiate TBI severity in the mild/moderate group when measured at 0h post-injury. Tau serum levels were increased in the mild/moderate group at 24h. In addition, NfL and p-tau181 showed increased serum levels at 48h in the aforementioned GCS category. Individual biomarker performance on predicting unfavorable outcomes was measured at 0, 24, and 48h across different GOS-E Peds time points, which was significant for p-tau181 at 0h at all time points, UCH-L1 at 0h at 6-9 months and 12 months, GFAP at 48h at 12 months, NfL at 0h at 12 months, tau at 0h at 12 months and S100B at 0h at 12 months. We concluded that TBI leads to increased serum neuronal injury biomarkers during the first 0-48h post-injury. A biomarker panel measuring these proteins could aid in the early diagnosis of mild to moderate pTBI and may predict neurological outcomes across the injury spectrum.

Objective: Even though all guidelines recommend generally against antipsychotic polypharmacy, antipsychotic polypharmacy appears to be a very common practice across the globe. This study aimed to examine the prescription patterns of antipsychotics in Qatar, in comparison with the international guidelines, and to scrutinize the sociodemographic and clinical features associated with antipsychotic polypharmacy., Methods: All the medical records of all the inpatients and outpatients treated by antipsychotics at the Department of Psychiatry-Hamad Medical Corporation (HMC) in Doha, Qatar (between October 2012 and April 2014) were retrospectively analyzed. We retrieved the available sociodemographic data, psychiatric features, and details on the medication history., Results: Our sample consisted of 537 individuals on antipsychotics (2/3 were male; mean age 33.8±10.2 years), prescribed for a psychotic disorder in 57%, a mood disorder in 9.3%, and various other diagnoses in 33.7%. About 55.9% received one antipsychotic, 29.6% received two antipsychotics, and 14.5% received more than two antipsychotics. Polypharmacy was associated with younger age (p = 0.025), being single (p<0.001), the diagnosis of a psychotic disorder (p<0.001), and previous admissions to psychiatry (p<0.001)., Conclusions: Antipsychotic polypharmacy appears to be quite common in Qatar, as it is the case in many other countries, in contrast with most international recommendations. Studies are needed to explore the reasons behind this disparity., Competing Interests: The authors have declared that no competing interests exist.

This study aimed to assess the differential effects of first-generation (FGA) and second-generation antipsychotics (SGA) on the prevalence of risk factors for metabolic syndrome among mentally ill patients in Qatar. We also wanted to check if there is proper adherence with the guidelines for prescribing antipsychotics and the monitoring of metabolic effects in this population. We collected the available retrospective data (socio-demographic, psychiatric, anthropometric, and metabolic measures) from the records of 439 patients maintained on antipsychotics. The majority were males, married, employed, having a psychotic disorder, and receiving SGA. Patients on SGA showed more obesity, higher BP, and more elevated triglycerides compared to those on FGA. The prevalence of the abnormal metabolic measures was high in this sample, but those on SGA showed a significantly higher prevalence of abnormal body mass index and BP. Obesity and hypertension were common in patients maintained on antipsychotics, especially those on SGA. Polypharmacy was common, and many metabolic measures were not monitored properly in those maintained on antipsychotics. More prospective studies with guided monitoring of the patients' clinical status and metabolic changes are needed to serve better this population of patients.

Objective: Fetal/neonatal alloimmune thrombocytopenia is a severe bleeding disorder, which can result in intracranial hemorrhage (ICH), leading to death or neurological sequelae. In whites, maternal anti-human platelet antigen-1a (HPA-1a) antibodies are responsible for the majority of cases. No predictive factors for ICH are available to guide prophylactic treatment during pregnancy. In this study, we investigated antibodies from mothers with ICH-positive fetal/neonatal alloimmune thrombocytopenia and with ICH-negative fetal/neonatal alloimmune thrombocytopenia to identify serological and functional differences between the groups., Approach and Results: In an antigen capture assay, we observed a stronger binding of +ICH antibodies to endothelial cell (EC)-derived αvβ3. By absorption experiments, we subsequently identified anti-HPA-1a antibodies of anti-αvβ3 specificity in the +ICH but not in the -ICH cohort. Only the anti-αvβ3 subtype, but not the anti-β3 subtype, induced EC apoptosis of HPA-1a-positive ECs by caspase-3/7 activation, and mediated by reactive oxygen species. In addition, only the anti-αvβ3 subtype, but not the anti-β3 subtype, interfered with EC adhesion to vitronectin and with EC tube formation., Conclusions: We conclude that the composition of the anti-HPA-1a antibody subtype(s) of the mother may determine whether ICH occurs. Analysis of anti-HPA-1a antibodies of the anti-αvβ3 subtype in maternal serum has potential in the diagnostic prediction of ICH development and may allow for modification of prophylactic treatment in fetal/neonatal alloimmune thrombocytopenia., (© 2016 American Heart Association, Inc.)

Cerebral ischemia is encompassed by cerebrovascular apoptosis, yet the mechanisms behind apoptosis regulation are not fully understood. We previously demonstrated inhibition of endothelial apoptosis by vascular endothelial growth factor (VEGF) through upregulation of poly(ADP-ribose)-polymerase (PARP) expression. However, PARP overactivation through oxidative stress can lead to necrosis. This study tested the hypothesis that neuropilin-1 (NP-1), an alternative VEGF receptor, regulates the response to cerebral ischemia by modulating PARP expression and, in turn, apoptosis inhibition by VEGF. In endothelial cell culture, NP-1 colocalized with VEGF receptor-2 (VEGFR-2) and acted as its coreceptor. This significantly enhanced VEGF-induced PARP mRNA and protein expression demonstrated by receptor-specific inhibitors and VEGF-A isoforms. NP-1 augmented the inhibitory effect of VEGF/VEGFR-2 interaction on apoptosis induced by adhesion inhibition through the αV-integrin inhibitor cRGDfV. NP-1/VEGFR-2 signal transduction involved JNK and Akt. In rat models of permanent and temporary middle cerebral artery occlusion, the ischemic cerebral hemispheres displayed endothelial and neuronal apoptosis next to increased endothelial NP-1 and VEGFR-2 expression compared to non-ischemic cerebral hemispheres, sham-operated or untreated controls. Increased vascular superoxide dismutase-1 and catalase expression as well as decreased glycogen reserves indicated oxidative stress in the ischemic brain. Of note, protein levels of intact PARP remained stable despite pro-apoptotic conditions through increased PARP mRNA production during cerebral ischemia. In conclusion, NP-1 is upregulated in conditions of imminent cerebrovascular apoptosis to reinforce apoptosis inhibition and modulate VEGF-dependent PARP expression and activation. We propose that NP-1 is a key modulator of VEGF maintaining cerebrovascular integrity during ischemia. Modulating the function of NP-1 to target PARP could help to prevent cellular damage in cerebrovascular disease., (© 2013.)

The osteoclast-associated receptor (OSCAR), primarily described as a co-stimulatory regulator of osteoclast differentiation, represents a potential link between bone metabolism and vascular biology. Previously, we identified OSCAR as an endothelial cell-derived target of the proatherogenic factor oxidized low density lipoprotein (oxLDL). Since monocytes play an important role in the progression of atherosclerosis, we assessed whether atherogenic stimuli also regulate the expression of OSCAR on monocytes. Four-week-old male wild-type (WT), apolipoprotein e knockout (apoe KO), and LDL receptor knockout (ldlr KO) mice were fed a high-fat diet or normal chow for 6weeks. Peripheral blood mononuclear cells (PBMCs) isolated from the spleen were stained with antibodies against CD14 and OSCAR for subsequent flow cytometric analysis. OSCAR surface expression on CD14-positive monocytes increased 2-fold in PBMCs from apoe KO mice compared to WT mice. Feeding a high-fat diet further increased OSCAR surface expression 1.5-fold in apoe KO mice compared to normal diet. Moreover, OSCAR-positive macrophages were detected in atherosclerotic plaques of apoe KO mice. Interestingly, monocytic OSCAR expression was not altered in ldlr KO mice. In the murine macrophage cell line RAW 264.7, TNFα and oxLDL induced OSCAR mRNA expression by 2-fold and 5-fold (p<0.01), respectively. Blocking the oxLDL receptor LOX-1 and inhibiting the NF-κB pathway prevented OSCAR induction. In conclusion, OSCAR expression in monocytic cells is regulated by proatherogenic stimuli further pointing towards a role in vascular inflammation or plaque vulnerability during atherosclerosis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Haemochromatosis may impair the function of endocrine organs, amongst others the pituitary gland. It was the aim of this study to determine pituitary function in adult patients with genetically defined hereditary haemochromatosis in a prospective diagnostic study using a standardised stimulation test. Therefore, 22 patients (7 females, 15 males; age at diagnosis of haemochromatosis 48.1 ± 7.9 years; age at study inclusion 50.7 ± 7.7 years) with genetically defined hereditary haemochromatosis were investigated by a combined pituitary stimulation test (CRH, GHRH/arginine, GnRH, TRH). In 11 patients (50% of the study population; 2 females, 9 males), pituitary insufficiencies were detected [isolated corticotrophic insufficiency (peak cortisol < 181.25 μg/l/500 nmol/l) n=10 (2 females, 8 males); combined corticotrophic and borderline gonadotrophic insufficiency (basal testosterone 2.4-3.0 μg/l without basal LH-elevation) in 1 male]. Somatotrophic pituitary insufficiencies were not found. IFG-1 concentrations below -2 standard deviations in 7 patients (32%) may be attributed to impaired hepatic IGF-1 synthesis. Hypopituitarism, particularly corticotrophic insufficiency, seems to be prevalent in a considerable number of middle-aged patients with hereditary haemochromatosis. Despite normal somatotrophic function, low IGF-1 serum concentrations may be found in a subgroup of haemochromatosis patients., (© Georg Thieme Verlag KG Stuttgart · New York.)

Objectives: Osteoprotegerin (OPG) is a regulator of bone and vascular homeostasis and acts as a decoy receptor for proapoptotic TNF-related apoptosis-inducing ligand (TRAIL)., Design and Methods: We assessed pericardial and serum levels of OPG and TRAIL in pericardial effusions (PE) of malignant (mPE, n=24) or non-malignant (nPE, n=34) origin, and in pericardial fluid (PF, n=25) of coronary artery disease (CAD) patients by ELISA., Results: OPG was at least 5 fold higher in PE or PF compared to serum, with a significantly higher ratio of pericardial to serum OPG in patients with mPE or nPE compared to PF (mPE vs. PF, p=0.011; nPE vs. PF, p<0.001). TRAIL was only detectable in mPE and PF. Logistic regression analysis revealed that a high ratio of pericardial to serum OPG and high TRAIL in PE were the best variable combination to predict malignancy of PE., Conclusions: Pericardial and systemic OPG or TRAIL are potential diagnostic tools to discriminate between malignant or benign PE., (Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Cross talks between the vascular and immune system play a critical role in vascular diseases, in particular in atherosclerosis. The osteoclast-associated receptor (OSCAR) is a regulator of osteoclast differentiation and dendritic cell maturation. Whether OSCAR plays a role in vascular biology and has an impact on atherogenic processes provoked by proinflammatory stimuli is yet unknown. We identified OSCAR on the surface of human primary endothelial cells. Stimulation of endothelial cells with oxidized low-density lipoprotein (oxLDL) caused a time- and dose-dependent induction of OSCAR, which was lectin-like oxidized LDL receptor 1 and Ca(2+) dependent. OSCAR was transcriptionally regulated by oxLDL as shown by OSCAR promoter analysis. Specific inhibition of the nuclear factor of activated T cells (NFAT) pathway prevented the oxLDL-mediated increase of endothelial OSCAR expression. As assessed by EMSA, oxLDL induced binding of NFATc1 to the OSCAR promoter. Notably, in vivo-modified LDL from patients with diabetes mellitus stimulated OSCAR mRNA expression in human endothelial cells. Furthermore, apolipoprotein E knockout mice fed a high-fat diet showed an enhanced aortic OSCAR expression associated with increased expression of NFATc1. In summary, OSCAR is expressed in vascular endothelial cells and is regulated by oxLDL involving NFATc1. Our data suggest that OSCAR, originally described in bone as immunological mediator and regulator of osteoclast differentiation, may be involved in cell activation and inflammation during atherosclerosis.

Background: Multiplexed cytokine measurement offers many advantages over the conventional enzyme-linked immunosorbent assay (ELISA) format when applied in large-scale epidemiological studies or clinical trials. In the present study we set out to define the reliability and consistency of a suspension multiplexed protein array, the cytometric bead array (CBA), in large-scale, longitudinal studies., Methods: The cytokines interleukin (IL)-5, IL-10, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were measured in pediatric samples from childhood asthma and allergy studies. Analytical performance of CBA was determined in sample supernatants and CBA was compared to conventional ELISA., Results: Within-run and total imprecision were between 5.2%-10.8% and 5.6%-13.2%, respectively, at three different concentrations for all cytokines. Slopes of dilution linearity were between 1.01 and 1.31 for the four cytokines. The recovery rate at two different concentrations of the cytokines was between 97% and 113%. Lower limits of detection and quantification as well as functional sensitivity were determined. Comparison of the multiplex array and solid phase method showed good correlation with r between 0.82 and 0.93. The sample volume required for the multiplex format was 25% of the ELISA sample volume., Conclusions: CBA analytical evaluation and comparison to an ELISA format demonstrated high reproducibility, sensitivity and good applicability for small volume samples.

Background: Vascular endothelial growth factor (VEGF) inhibits the endothelial apoptosis that is induced by caspases during vascular remodeling; however, the underlying mechanisms have not been completely elucidated., Objectives: We hypothesized that VEGF upregulates poly(ADP-ribose) polymerase-1 (PARP) as a caspase mediator, and sought to investigate the link between apoptosis inhibition by VEGF and PARP regulation in the human vasculature., Methods: Human endothelial cells (primary cells, macrovascular/microvascular lines) were incubated with 100 pg mL(-1) to 1 μg mL(-1) VEGF-A(165) in the absence or presence of PARP small interfering RNA (siRNA). Apoptosis induced by integrin inhibition was measured by flow cytometry, trypan blue exclusion, and nuclear morphology. PARP expression and activity were determined by real-time RT-PCR, Western blot, and ribosylation assay. VEGF receptors and signal transduction were analyzed by inhibitor experiments, enzyme assays, western blot, and immunofluorescence microscopy. Immunohistochemistry was applied to a vascular culture model and to 24 atherosclerotic and 10 normal human arteries., Results: VEGF-A(165) induced resistance to apoptosis caused by caspase activation in endothelial cells in a time-dependent manner. VEGF, but not fibroblast growth factor-2 or transforming growth factor-β, time-dependently and dose-dependently induced PARP expression and activity, involving VEGF receptor-2 colocalized with neuropilin-1 as well as the signal transduction molecules c-Jun N-terminal kinase and Akt. The antiapoptotic effect of VEGF was abrogated by PARP siRNA. The relationship between local VEGF influence and endothelial PARP expression was confirmed in human arteries and the vascular culture model., Conclusions:  VEGF exerts its antiapoptotic effect on the endothelium through the regulation of PARP expression. PARP has been attributed an ambiguous role in apoptosis; here, we show that PARP promotes vascular endothelial integrity in VEGF-associated processes., (© 2011 International Society on Thrombosis and Haemostasis.)

The term osteoimmunology is coined for molecular and cellular cross talk between the skeletal and immune system. Immunomodulatory signals have long been implicated as key regulators of bone metabolism. Recently, osteoclast-associated receptor (OSCAR), an IgG-like receptor, has been identified as an important osteoimmunological mediator. OSCAR expression in bone is highly conserved across different species, and the molecule is an important costimulatory receptor for osteoclast differentiation through activation of NFATc1. In humans, OSCAR is expressed by macrophages, monocytes, and monocyte-derived dendritic cells and modulates the response of the innate and adaptive immune systems by promoting cell activation and maturation, Ag presentation, and proinflammatory circuits. Human studies indicate that OSCAR may contribute to the pathogenesis and severity of osteoporosis and rheumatoid arthritis. In this paper, we review the structure-function relationship, expression pattern, and physiological role of OSCAR in osteoimmunology and summarize its potential implications for human diseases.

Background: There is evidence that selenium levels are relatively low in Europe and may be falling. Low levels of selenium or low activity of some of the enzymes dependent on selenium have been associated with asthma., Methods: The GA(2)LEN network has organized a multicentre case-control study in Europe to assess the relation of plasma selenium to asthma. The network compared 569 cases in 14 European centres with a diagnosis of asthma and reporting asthma symptoms in the last 12 months with 576 controls from the same centres with no diagnosis of asthma and no asthmatic symptoms in the last 12 months., Results: All cases and controls were selected from the same population defined by age and place of residence. Mean plasma selenium concentrations among the controls ranged from 116.3 microg/l in Palermo to 67.7 microg/l in Vienna and 56.1 microg/l among the children in Oslo. Random effects meta-analysis of the results from the centres showed no overall association between asthma and plasma selenium [odds ratio (OR)/10 microg/l increase in plasma selenium: 1.04; 95% confidence interval (CI): 0.89-1.21] though there was a significantly protective effect in Lodz (OR: 0.48; 95% CI: 0.29-0.78) and a marginally significant adverse effect in Amsterdam (OR: 1.68; 95% CI: 0.98-2.90) and Ghent (OR: 1.35; 95% CI: 1.03-1.77)., Conclusion: This study does not support a role for selenium in protection against asthma, but effect modification and confounding cannot be ruled out.

Dendritic cells (DC) comprise a unique leukocyte population which controls primary immune responses. Recent studies indicate that DC express osteoprotegerin (OPG), a secreted tumor necrosis factor receptor homolog, which regulates DC survival, monocyte chemotaxis, and B cell development and function by ligating TNF family member receptor activator of NF-kappaB ligand (RANKL). The precise regulators of OPG expression in DC have not been investigated. In this study, we assessed OPG mRNA steady state levels by Northern blot analysis and OPG protein secretion by an immunoassay in monocyte-derived DC of different maturation, and the effect of different cytokines and hormones on OPG expression. OPG was upregulated with maturation of DC, whereas pretreatment of DC with 1alpha,25(OH)(2) vitamin D(3), tamoxifen, or dexamethasone, agents that inhibit differentiation of DC, decreased OPG expression. In vivo, OPG was found to be colocalized with mature CD83(+) DC in human tonsils by immunofluorescence confocal microscopy analysis. Furthermore, OPG was upregulated by TNF superfamily members TNF-alpha, anti-CD40, and RANKL, and by ligands of the Toll-like/IL-1 receptor family including IL-1beta, double-stranded RNA (poly I:C), or lipopolysaccharide (LPS), all of which induce maturation of DC. Gene silencing by small interfering RNA (siRNA) directed against transcription factor NF-kappaB abrogated the expression of OPG as demonstrated by real-time PCR. In summary, we describe that the expression of OPG by DC increases with maturation and is NF-kappaB-dependent, possibly regulating immune responses in lymphoid tissues.

Lipoprotein(a) [Lp(a)], consisting of LDL and the unique constituent apolipoprotein(a) [apo(a)], which contains multiple repeats resembling plasminogen kringle 4, is considered a risk factor for the development of atherosclerotic disorders. However, the underlying mechanisms for the atherogenicity of Lp(a) are not completely understood. Here, we define a novel function of Lp(a) in promoting inflammatory cell recruitment that may contribute to its atherogenicity. Through its apo(a) moiety Lp(a) specifically interacts with the beta2-integrin Mac-1, thereby promoting the adhesion of monocytes and their transendothelial migration in a Mac-1-dependent manner. Interestingly, the interaction between Mac-1 and Lp(a) was strengthened in the presence of proatherogenic homocysteine and was blocked by plasminogen/angiostatin kringle 4. Through its interaction with Mac-1, Lp(a) induced activation of the proinflammatory transcription factor NFkappaB, as well as the NFkappaB-related expression of prothrombotic tissue factor. In atherosclerotic coronary arteries Lp(a) was found to be localized in close proximity to Mac-1 on infiltrating mononuclear cells. Taken together, our data demonstrate that Lp(a), via its apo(a) moiety, is a ligand for the beta2-integrin Mac-1, thereby facilitating inflammatory cell recruitment to atherosclerotic plaques. These observations suggest a novel mechanism for the atherogenic properties of Lp(a).

Apoptosis has been attributed an essential role in dilated cardiomyopathy (DCM) recently. We assessed expression of TNF-related apoptosis-inducing ligand (TRAIL) and its decoy receptor osteoprotegerin (OPG) in men with nonischemic DCM, who underwent coronary angiography and endomyocardial biopsy (EMB) after exclusion of coronary artery disease compared to control patients. TRAIL plasma concentrations were elevated in DCM (p=0.02 vs. controls), and were positively correlated with left ventricular enddiastolic diameter (r=0.15, p=0.04), whereas OPG plasma levels did not differ between both groups (p=0.96). In EMB of DCM patients, TRAIL and OPG protein were detected by immunohistochemistry but not in controls. Furthermore, gene expression in EMB or peripheral blood leukocytes (PBL) of DCM patients assessed by real-time PCR showed an increase of TRAIL mRNA in PBL (p=0.01 vs. controls), whereas OPG mRNA was upregulated in endomyocardial specimens (p<0.001 vs. controls). In conclusion, myocardial overexpression of antiapoptotic OPG in DCM patients may represent a compensatory mechanism to limit systemic activation of TRAIL in patients with congestive heart disease.

The junctional adhesion molecule-C (JAM-C) was recently shown to be a counter receptor for the leukocyte beta2-integrin Mac-1 (CD11b/CD18), thereby mediating interactions between vascular cells, particularly in inflammatory cell recruitment. Here, we investigated the role of JAM-C in oxidized low-density lipoprotein (LDL)-mediated leukocyte recruitment. As compared with normal arteries, immunostaining of atherosclerotic vessels revealed a high expression of JAM-C in association with neointimal smooth muscle cells and the endothelium. Moreover, JAM-C was strongly up-regulated in the spontaneous early lesions in ApoE -/- mice. In vitro, cultured human arterial smooth muscle cells (HASMC) were found to express JAM-C, and oxLDL, as well as enzymatically modified LDL (eLDL) significantly up-regulated JAM-C on both HASMC and endothelial cells in a time- and dose-dependent manner. Although under quiescent conditions, JAM-C predominantly localized to interendothelial cell-cell contacts in close proximity to zonula occludens-1 (ZO-1), oxLDL treatment induced a disorganization of JAM-C localization that was no more restricted to the interendothelial junctions. JAM-C thereby mediated both leukocyte adhesion and leukocyte transendothelial migration upon oxLDL treatment of endothelial cells, whereas JAM-C on quiescent endothelial cells only mediates leukocyte transmigration. Thus, upon oxLDL stimulation endothelial JAM-C functions as both an adhesion, as well as a transmigration receptor for leukocytes. Taken together, JAM-C is up-regulated by oxLDL and may thereby contribute to increased inflammatory cell recruitment during atherosclerosis. JAM-C may therefore provide a novel molecular target for antagonizing interactions between vascular cells in atherosclerosis.

Vascular calcification may occur at different areas of the vessel wall, including the intima in atherosclerosis and the media in Mönckeberg's sclerosis. Medial calcification of arteries is common in patients with diabetes mellitus or chronic renal failure. Osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand are essential modulators of bone homeostasis and may be involved in the process of vascular calcification. In this study we investigated arteries from patients with Mönckeberg's sclerosis and atherosclerosis. Apoptosis, which precedes vascular calcification in vitro, was assessed by an in situ ligation assay and was localized to the medial layer of arteries (Mönckeberg's sclerosis) and the neointima (atherosclerosis). Immunohistochemistry and in situ hybridization revealed OPG immunoreactivity and mRNA expression surrounding calcified areas in the medial layer (Mönckeberg's sclerosis), whereas OPG was mainly expressed adjacent to calcified neointimal lesions (atherosclerosis). Receptor activator of nuclear factor-kappaB ligand protein and mRNA were barely or not detectable. Of note, TNF-related apoptosis-inducing ligand, an inducer of apoptosis that is also blocked by OPG, displayed a similar spatial distribution as OPG. In summary, we demonstrate enhanced apoptosis adjacent to vascular calcification, and the concurrent expression of regulators of apoptosis and osteoclastic differentiation, TNF-related apoptosis-inducing ligand and OPG, suggesting their involvement in the pathogenesis of vascular calcification.

The factor VII activating protease (FSAP) is a serine-protease present in human plasma that serves to activate single-chain plasminogen activators, as well as coagulation factor VII. FSAP was localized within atherosclerotic lesions, and a genetic polymorphism in FSAP is associated with carotid stenosis. Hence, this study was conducted to gain broader insights into the cellular effects of FSAP on vascular smooth muscle cells (VSMC). DNA synthesis and cell proliferation assays revealed an inhibitory action of FSAP on platelet-derived growth factor BB (PDGF-BB)-mediated proliferation of VSMC. FSAP also inhibited PDGF-BB-induced migration of VSMC. These cellular effects of FSAP could be neutralized by an anti-FSAP mAb as well as by protease inhibitors such as aprotinin or a chloromethylketone inhibitor. Moreover, unfractionated heparin promoted the antiproliferative effect of FSAP on VSMC and was essential for the inhibition of VSMC migration. FSAP inhibited PDGF-BB binding to human VSMC and concomitantly blocked PDGF-BB-dependent phosphorylation of mitogen activated protein kinase p42/p44 and tyrosine phosphorylation of other proteins. These results unravel a new function of FSAP as an inhibitor of the proatherogenic phenotype of vascular smooth muscle.

Protease nexin-1 (PN-1) and plasminogen activator inhibitor-1 (PAI-1) are serine protease inhibitors that bind to the extracellular matrix protein vitronectin (VN) with high affinity. PAI-1 is known to inhibit cell adhesion and migration by binding to VN and inhibiting the interaction with integrins or the urokinase receptor (uPAR). Unexpectedly, PN-1 was found to increase the association between VN and uPAR in the presence of enzymatically active uPA. Through this mechanism PN-1 also stimulated uPAR-dependent cell adhesion to immobilized VN. In contrast to PAI-1, PN-1 did not influence VN binding to integrins or integrin-mediated cell adhesion. Upon adhesion of monocytes to VN there was an accumulation of uPAR and PN-1 at the interface between the cell and the matrix, whereas on fibronectin (FN) both components were distributed evenly over the whole cell as visualized by confocal microscopy. Immunohistochemistry of atherosclerotic vessels indicated that PN-1 was found associated with smooth muscle cells, macrophages and platelets. In some regions of the diseased vessels PN-1 was in close proximity to VN and uPAR, but no PN-1 was present in normal vessels. These results indicate a novel function of PN-1 linked to complex formation with uPA that leads to the regulation of VN-dependent adhesion of leukocytes.

The pattern recognition receptor, RAGE (receptor for advanced glycation endproducts), propagates cellular dysfunction in several inflammatory disorders and diabetes. Here we show that RAGE functions as an endothelial adhesion receptor promoting leukocyte recruitment. In an animal model of thioglycollate-induced acute peritonitis, leukocyte recruitment was significantly impaired in RAGE-deficient mice as opposed to wild-type mice. In diabetic wild-type mice we observed enhanced leukocyte recruitment to the inflamed peritoneum as compared with nondiabetic wild-type mice; this phenomenon was attributed to RAGE as it was abrogated in the presence of soluble RAGE and was absent in diabetic RAGE-deficient mice. In vitro, RAGE-dependent leukocyte adhesion to endothelial cells was mediated by a direct interaction of RAGE with the beta2-integrin Mac-1 and, to a lower extent, with p150,95 but not with LFA-1 or with beta1-integrins. The RAGE-Mac-1 interaction was augmented by the proinflammatory RAGE-ligand, S100-protein. These results were corroborated by analysis of cells transfected with different heterodimeric beta2-integrins, by using RAGE-transfected cells, and by using purified proteins. The RAGE-Mac-1 interaction defines a novel pathway of leukocyte recruitment relevant in inflammatory disorders associated with increased RAGE expression, such as in diabetes, and could provide the basis for the development of novel therapeutic applications.

Regulation of alphavbeta3 and alpha5beta1 integrin function plays a crucial role in atherosclerosis. Possible regulators of integrin-matrix interactions are integrin-binding ADAMs (proteins with a disintegrin- and metalloproteinase-domain), like ADAM-15 and ADAM-9. Molecular interactions between ADAM-15, alpha5beta1, and alphavbeta3 have been demonstrated. ADAM-9 and ADAM-15 were found to be interdependently regulated. This study, therefore, investigated whether the upregulation of integrins alpha5beta1 and alphavbeta3 was correlated with the expression of integrin-binding ADAMs in atherosclerotic processes. Human arterial and venous vascular smooth muscle cells (VSMCs) were incubated with PDGF over different time intervals up to a 3-day culture period. mRNA concentrations, quantified by real-time RT-PCR and normalized to PBGD, of integrins alphavbeta3 and alpha5beta1 were strongly increased after a 12-h PDGF-incubation in arterial and venous VSMC. ADAM-15 and ADAM-9 mRNA production showed a corresponding increase following integrin upregulation after a 24-h incubation period. Western blot anaylsis revealed an increased protein expression of integrins and ADAMs in PDGF-stimulated VSMC. Additionally, mRNA concentrations of atherosclerotic and normal human specimens were quantified by real-time RT-PCR. mRNA of ADAMs and integrins was significantly increased in atherosclerotic arteries compared to normal arteries. Immunohistochemistry of these specimens showed an increased expression and codistribution of both ADAMs and integrins in atherosclerosis. In conclusion, upregulation of ADAM-15 and ADAM-9 in atherosclerosis appears to follow an increase in alpha5beta1 and alphavbeta3 integrins. Since alpha5beta1 and alphavbeta3 are known to promote smooth muscle cell migration and proliferation, upregulation of ADAM-15 and ADAM-9 could balance integrin-matrix interactions and cell migration, thus modulating neointima progression., (Copyright 2003 Wiley-Liss, Inc.)

Plasminogen activator inhibitor-1 (PAI-1) and two-chain high molecular weight kininogen (HKa) exert anti-adhesive properties in vitronectin-dependent cell adhesion. Here, the hypothesis was tested that these anti-adhesive components promote apoptosis in vascular cells. PAI-1 or HKa induced a 2- to 3-fold increase in apoptosis of human umbilical-vein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC) adherent to vitronectin, as determined by annexin V-FACS assay, similar to alphav-integrin inhibitor cyclo-(Arg-Gly-Asp-D-Phe-Val)-peptide (cRGDfV). Apoptosis occurred after 12 h incubation and was attributable to caspase 3 activation that in turn induced DNA fragmentation. Induction of apoptosis strongly correlated with the anti-adhesive effect of PAI-1 and HKa on these cells. In contrast, PAI-1 and HKa did not affect fibronectin-dependent adhesion or cell survival. uPA did not influence apoptosis in vitronectin- or fibronectin-adherent cells. In atherosclerotic vessel sections, congruent distribution of vitronectin, PAI-1, HK, and of components of the urokinase plasminogen activator/receptor system with apoptotic cells lining foam cell lesions was demonstrated by immunostaining. These results indicate that inhibition of vitronectin-dependent cell adhesion through PAI-1 and HKa correlates with apoptosis induction in vascular cells mediated through the caspase 3 pathway. Co-distribution of apoptosis with plasminogen activation system components in atherosclerosis exemplifies the significance of anti-adhesive mechanisms and apoptosis for tissue remodeling, such as in neointima development.

The objective was to investigate whether the renin-angiotensin (RA) system and related peptides endothelin-1 (ET-1) and vasopressin (VP) influence the development of coronary artery disease (CAD). Angiotensin I-converting enzyme (ACE) insertion/deletion (I/D) gene polymorphism has been associated with the risk of CAD. The ACE I/D polymorphism determines ACE activity, but plasma levels of other RA system components remain unchanged. However, ET-1 and VP production could be increased by RA system-dependent stimulation, continually promoted by paracrine stimulation and sustained by neointimal growth. ET-1 and VP have not been associated with the ACE I/D polymorphism so far. The present study investigated the association of the ACE I/D polymorphism with plasma concentrations of ET-1 and VP, as well as with renin, angiotensin-II (AT-II) and ACE activity in 98 Caucasian individuals with CAD. ACE I/D polymorphism showed no association with plasma levels of VP, ET-1, AT-II or renin. These parameters were also not associated taking into consideration different patient variables, such as diabetes mellitus, hypertension or severity of CAD. Only plasma ACE activity was associated with the D allele. In conclusion, the ACE I/D polymorphism could not be related to plasma concentrations of VP, ET-1, renin or AT-II, but as previously demonstrated, it could only be related to ACE activity in patients with CAD. Differences in ACE activity between ACE I/D genotype subgroups are probably compensated within the RA system itself or within non-ACE pathways, so that plasma concentrations of the related peptides ET-1 and VP remain unaffected.

Objective: In arteriosclerosis and bypass graft stenosis, intimal proliferation is controlled by local and systemic growth factors, such as platelet derived growth factor (PDGF) or insulin. Intimal hyperplasia can be produced in organ culture models. Our aim was to compare neointima formation in two organ culture models of internal mammary artery (IMA) and saphenous vein (SV), with special reference to the influence of systemic and local growth stimuli., Methods: Rings of freshly isolated human SV and IMA were cultured over a 3-, 6- or 8-day period. They were distributed into five groups of incubation protocols: incubation with 10% serum; insulin 50 ng/ml and 100 ng/ml; PDGF-BB 5 ng/ml and 10 ng/ml. Frozen sections of cultured rings and pre-culture segments were subjected to elastic stain and immunohistochemistry. Antibodies directed against beta-actin and smooth muscle alpha-actin were used to characterize smooth muscle cell phenotype and against proliferating cell nuclear antigen (PCNA) to demonstrate proliferating cells., Results: Growth factor incubation caused massive intimal hyperplasia with increased elastic fibers in SV and intimal smooth muscle cell as well as matrix accumulation in IMA. Intimal thickening, PCNA and beta-actin expression reached their maximum on day 6 of culture. In both culture models, serum, insulin and PDGF caused increasing intimal thickening, with more pronounced effects in SV., Conclusions: These organ culture models demonstrate the effects of insulin and PDGF on intimal hyperplasia in IMA and SV representing models for arteriosclerosis and bypass graft stenosis and stressing the role of insulin and growth factors for neointima development.

Different extracellular matrix proteins have been described as binding proteins for growth factors, influencing their storage or presentation towards cellular receptors. The multifunctional adhesive glycoprotein vitronectin (VN), which is found in the circulation and widely distributed throughout different tissues, has been implicated in the regulation of vascular cell functions, and these activities could be related to interactions with various growth factors. In vitro, soluble VN interfered with transforming growth factor-beta (TGF-beta) binding to isolated extracellular matrix and was found to associate with TGF-beta1 and TGF-beta2 as well as with other growth factors such as vascular endothelial growth factor, epidermal growth factor, or basic fibroblast growth factor in a saturable manner. In particular, binding of TGF-beta was maximal for the heparin-binding multimeric isoform of VN, whereas VN in a ternary complex with thrombin and antithrombin or plasma VN exhibited weaker binding. Plasminogen activator inhibitor-1 (PAI-1) or heparin, but not desulfated glycosaminoglycans, interfered with binding of VN to TGF-beta, and soluble PAI-1 was able to dissociate VN-bound TGF-beta. Upon limited plasmin proteolysis of VN, only the fragments comprising the intact aminoterminal portion of VN bound to TGF-beta as did a synthetic peptide (amino acids 43 to 62), indicating that TGF-beta and PAI-1 share common binding site(s) on VN. Although VN did not influence TGF-beta bioactivity for mink lung epithelial cells, TGF-beta dose dependently inhibited both urokinase-receptor as well as alpha(v)-integrin-dependent adhesion to VN. This activity of TGF-beta was reminiscent of the antiadhesive function of PAI-1. In atherosclerotic tissue sections, staining patterns of VN and TGF-beta indicated their colocalization. These findings describe VN as a new binding protein for TGF-beta, whereby specific functions of both factors become modulated by this interaction.

The problem of T classification of proximal gastric carcinomas is becoming increasingly important due to a rise in the incidence of these tumors. The aim of this study was to examine the gastric insertion of the lesser and greater omenta and its role in the T classification of gastric carcinomas. The stomach and greater and lesser omenta were removed from 76 fixed cadavers and 12 measurements each were done in defined localizations. The lesser omentum extended to the gastric wall in 98% of the cases. This junction as well as the omental thickness and thus the retroperitoneal part are especially pronounced in the cardiac region. According to the current UICC classification, even advanced tumors extending into the gastric wall can be classified T2 as long as they do not penetrate the visceral peritoneum. This results in "understaging" and a seemingly poorer prognosis for cardiac carcinomas. Our study results support the recommendation of Hermanek and Wittekind [5] to subdivide the T2 stage of gastric carcinomas on the basis of infiltration depth.

Objective: The influence of the operation technique on the complication rate of thyroid operations is still a subject of discussions. In this study, we analysed therefore the most important complications, i.e. palsy of the recurrent laryngeal nerve and hypoparathyroidism, specially under the aspect of routine exposition of the recurrent laryngeal nerve and parathyroids., Methods: 116 patients with goiter operations of the year 1995 (12 different surgeons) were analysed. Age of patients was between 7 and 72 years, mean age 45 years. The operations performed were 70 subtotal resections (benign goiter), 33 near-total-resections (diffuse autonomy or Graves' disease), 33 hemithyroidectomies (scintigraphically "cold" nodules), 4 adenoma resections with combinations of these resections in 24 cases., Results: 4.3% of the patients (5/116 patients) developed an unilateral recurrent nerve palsy immediately after operation. Persisting recurrent nerve palsy occurred only in 1.7% (2/116) of these cases. Postoperative nerve palsy was mainly found after extensive resections (2/33 near-total-resections) or operations of recurrent goiter (2/6 re-operations), rarely after routine operations (1/70 subtotal resections). Hypocalcemia was found in 9.5% of the patients (11/116), of which 9 cases were asymptomatic (7.8%) and 2 symptomatic by tetany (1.7%). Persisting hypocalcemia for longer than one week was found in 3.5% (4/116), permanent hypoparathyroidism in 1.7% (2/116) of the patients., Conclusions: Routine systematic exposure of recurrent laryngeal nerves and parathyroids reduces the incidence of complications in thyroid surgery to a minimum. The risk of recurrent nerve palsy increases with extent and difficulty of the operation, with the highest risk for nerve lesions seen in re-operations and near-total-resections.

During the development of somites in mouse embryos, widespread activity of unspecific cholinesterase (BuChE) was demonstrated after prolonged incubation. Independent of their position, all somite cells and their derivatives (dermatome, myotome and sclerotome) exhibited enzyme activity in the perinuclear space and in the endoplasmic reticulum. The plasmalemma did not show any enzyme activity. Differentiation of the sclerotome into vertebrae was accompanied by a reduction of BuChE. However, a low enzyme reaction was still present in the first typical differentiated chondroblasts. Notochordal cells were detectable by their high BuChE content. This was also found in cells already showed severe degeneration. In addition to BuChE, acetylcholinesterase (AChE) was first visible of day 9 of embryonic development in newly formed myotubes of the myotomes. Some hypotheses on the functional significance of of embryonic BuChE are discussed in the light of these results.