Enhanced activity of meprin-?, a pro-migratory and pro-angiogenic protease, in colorectal cancer

Meprin-α is a metalloprotease overexpressed in cancer cells, leading to the accumulation of this protease in a subset of colorectal tumors. The impact of increased meprin-α levels on tumor progression is not known. We investigated the effect of this protease on cell migration and angiogenesis in vitro and studied the expression of meprin-α mRNA, protein and proteolytic activity in primary tumors at progressive stages and in liver metastases of patients with colorectal cancer, as well as inhibitory activity towards meprin-α in sera of cancer patient as compared to healthy controls. We found that the hepatocyte growth factor (HGF)-induced migratory response of meprin-transfected epithelial cells was increased compared to wild-type cells in the presence of plasminogen, and that the angiogenic response in organ-cultured rat aortic explants was enhanced in the presence of exogenous human meprin-α. In patients, meprin-α mRNA was expressed in colonic adenomas, primary tumors UICC (International Union Against Cancer) stage I, II, III and IV, as well as in liver metastases. In contrast, the corresponding protein accumulated only in primary tumors and liver metastases, but not in adenomas. However, liver metastases lacked meprin-α activity despite increased expression of the corresponding protein, which correlated with inefficient zymogen activation. Sera from cancer patients exhibited reduced meprin-α inhibition compared to healthy controls. In conclusion, meprin-α activity is regulated differently in primary tumors and metastases, leading to high proteolytic activity in primary tumors and low activity in liver metastases. By virtue of its pro-migratory and pro-angiogenic activity, meprin-α may promote tumor progression in colorectal cancer.

Role of KCNMA1 in breast cancer

KCNMA1 encodes the α-subunit of the large conductance, voltage and Ca(2+)-activated (BK) potassium channel and has been reported as a target gene of genomic amplification at 10q22 in prostate cancer. To investigate the prevalence of the amplification in other human cancers, the copy number of KCNMA1 was analyzed by fluorescence-in-situ-hybridization (FISH) in 2,445 tumors across 118 different tumor types. Amplification of KCNMA1 was restricted to a small but distinct fraction of breast, ovarian and endometrial cancer with the highest prevalence in invasive ductal breast cancers and serous carcinoma of ovary and endometrium (3-7%). We performed an extensive analysis on breast cancer tissue microarrays (TMA) of 1,200 tumors linked to prognosis. KCNMA1 amplification was significantly associated with high tumor stage, high grade, high tumor cell proliferation, and poor prognosis. Immunofluorescence revealed moderate or strong KCNMA1 protein expression in 8 out of 9 human breast cancers and in the breast cancer cell line MFM223. KCNMA1-function in breast cancer cell lines was confirmed by whole-cell patch clamp recordings and proliferation assays, using siRNA-knockdown, BK channel activators such as 17ß-estradiol and the BK-channel blocker paxilline. Our findings revealed that enhanced expression of KCNMA1 correlates with and contributes to high proliferation rate and malignancy of breast cancer.

Effects of Aerobic Fitness and Adiposity on Coagulation Biomarkers in Men vs. Women with Elevated Blood Pressure

A hypercoagulable state is a potential mechanism linking elevated blood pressure (BP), adiposity and a sedentary lifestyle to development of coronary heart disease (CHD). We examined relationships among aerobic fitness and adiposity in 76 sedentary subjects with elevated BP. Blood levels of plasminogen activator inhibitor-1 (PAI-1), D-dimer, von Willebrand factor (vWF) and thrombomodulin were assessed as biomarkers of coagulation. In individuals with elevated BP, percent body fat and fitness were associated with biomarkers indicative of a hypercoagulable state, even after demographic and metabolic factors were considered. D-dimer was positively associated with percent body fat (beta=0.37, p=0.003). PAI-1 was higher in men than in women (beta=-0.31, p=0.015) and associated with lower VO2peak (beta=-0.35, p=0.024). Thrombomodulin was positively associated with VO2peak (beta=0.56, p< 0.01). vWF was not significantly associated with fitness or adiposity. Our results emphasise that both percent body fat and physical fitness are important in the maintenance of haemostatic balance.

EFFETS DE L’INCORPORATION D’ADJUVANTS MINÉRAUX SUR LES PROPRIÉTÉS DE CIMENTS GÉOPOLYMÈRES A BASE DE SCORIES VOLCANIQUES

Volcanic ashes are raw materials from geological deposits with a range of chemical compositions. When combined with suitable alkali activators they can be converted to geopolymers cement at ambient temperature. In this work we have investigated the possibility of use bauxite and oyster shells as mineral admixture in volcanic ashes, to enhance the properties of geopolymers synthesized. Different methods of analyses such as Fourier Transform Infrared spectroscopy (FTIR), X-ray diffractometry (XRD), and Scanning Electron Microscopy (SEM) were used to assess the variation of setting time, linear shrinkage and 28 days compressive strength of geopolymers paste. The bauxite and the oyster shells were characterized using inductively coupled plasma (ICP-AES), thermal analyses (DSC/ATG), FTIR and X-ray diffractometry. The results of these analyses has showed that bauxite and oyster shells are respectively source of Al2O3 and of CaO, and can compensate the deficiencies of these oxides in volcanic ashes. Adding mineral admixture dissolve slowly in high alkaline medium. Addition of about 20% of bauxite or 10% of oyster shells is seen to decrease the setting time respectively from 415 to 275 min and 195 min. Linear shrinkage decrease with percentage of bauxite or of oyster shells added. Efflorescence is reduced by adding 10% of bauxite. 28 days compressive strength of geopolymeric materials increase respectively for 4.77 and 7.52% for 10% of bauxite or 20% of oyster shells added. More than these percentage additive has a deleterious effect on compressive strength due to crystalized mineralogical phases of the admixture.

Role of RhoA/ROCK-dependent actin contractility in the induction of tenascin-C by cyclic tensile strain

In chick embryo fibroblasts, the mRNA for extracellular matrix protein tenascin-C is induced 2-fold by cyclic strain (10%, 0.3 Hz, 6 h). This response is attenuated by inhibiting Rho-dependent kinase (ROCK). The RhoA/ROCK signaling pathway is primarily involved in actin dynamics. Here, we demonstrate its crucial importance in regulating tenascin-C expression. Cyclic strain stimulated RhoA activation and induced fibroblast contraction. Chemical activators of RhoA synergistically enhanced the effects of cyclic strain on cell contractility. Interestingly, tenascin-C mRNA levels perfectly matched the extent of RhoA/ROCK-mediated actin contraction. First, RhoA activation by thrombin, lysophosphatidic acid, or colchicine induced tenascin-C mRNA to a similar extent as strain. Second, RhoA activating drugs in combination with cyclic strain caused a super-induction (4- to 5-fold) of tenascin-C mRNA, which was again suppressed by ROCK inhibition. Third, disruption of the actin cytoskeleton with latrunculin A abolished induction of tenascin-C mRNA by chemical RhoA activators in combination with cyclic strain. Lastly, we found that myosin II activity is required for tenascin-C induction by cyclic strain. We conclude that RhoA/ROCK-controlled actin contractility has a mechanosensory function in fibroblasts that correlates directly with tenascin-C gene expression. Previous RhoA/ROCK activation, either by chemical or mechanical signals, might render fibroblasts more sensitive to external tensile stress, e.g., during wound healing.

Histamine increases sphingosine kinase-1 expression and activity in the human arterial endothelial cell line EA.hy 926 by a PKC-alpha-dependent mechanism

Sphingosine 1-phosphate (S1P) is a potent mitogenic signal generated from sphingosine by the action of sphingosine kinases (SKs). In this study, we show that in the human arterial endothelial cell line EA.hy 926 histamine induces a time-dependent upregulation of the SK-1 mRNA and protein expression which is followed by increased SK-1 activity. A similar upregulation of SK-1 is also observed with the direct protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, SK-2 activity is not affected by neither histamine nor TPA. The increased SK-1 protein expression is due to stimulated de novo synthesis since cycloheximide inhibited the delayed SK-1 protein upregulation. Moreover, the increased SK-1 mRNA expression results from an increased promoter activation by histamine and TPA. In mechanistic terms, the transcriptional upregulation of SK-1 is dependent on PKC and the extracellular signal-regulated protein kinase (ERK) cascade since staurosporine and the MEK inhibitor U0126 abolish the TPA-induced SK-1 induction. Furthermore, the histamine effect is abolished by the H1-receptor antagonist diphenhydramine, but not by the H2-receptor antagonist cimetidine. Parallel to the induction of SK-1, histamine and TPA stimulate an increased migration of endothelial cells, which is prevented by depletion of the SK-1 by small interfering RNA (siRNA). To appoint this specific cell response to a specific PKC isoenzyme, siRNA of PKC-alpha, -delta, and -epsilon were used to selectively downregulate the respective isoforms. Interestingly, only depletion of PKC-alpha leads to a complete loss of TPA- and histamine-triggered SK-1 induction and cell migration. In summary, these data show that PKC-alpha activation in endothelial cells by histamine-activated H1-receptors, or by direct PKC activators leads to a sustained upregulation of the SK-1 protein expression and activity which, in turn, is critically involved in the mechanism of endothelial cell migration.

Nitric oxide down-regulates the expression of the catalytic NADPH oxidase subunit Nox1 in rat renal mesangial cells

Glomerular mesangial cells can produce high amounts of nitric oxide (NO) and reactive oxygen species (ROS). Here we analyzed the impact of NO on the ROS-generating system, particularly on the NADPH oxidase Nox1. Nox1 mRNA and protein levels were markedly decreased by treatment of mesangial cells with the NO-releasing compound DETA-NO in a concentration- and time-dependent fashion. By altering the cGMP signaling system with different inhibitors or activators, we revealed that the effect of NO on Nox1 expression is at least in part mediated by cGMP. Analysis of a reporter construct comprising the 2547 bp of the nox1 promoter region revealed that a stimulatory effect of IL-1beta on nox1 transcription is counteracted by an inhibitory effect of IL-1beta-evoked endogenous NO formation. Moreover, pretreatment of mesangial cells with DETA-NO attenuated platelet-derived growth factor (PDGF)-BB or serum stimulated production of superoxide as assessed by real-time EPR spectroscopy and dichlorofluorescein formation. Transfection of mesangial cells with siRNAs directed against Nox1 and Nox4 revealed that inhibition of Nox1, but not Nox4 expression, is responsible for the reduced ROS formation by NO. Obviously, there exists a fine-tuned crosstalk between NO and ROS generating systems in the course of inflammatory diseases.

Association between sleep apnea severity and blood coagulability: Treatment effects of nasal continuous positive airway pressure

A prothrombotic state may contribute to the elevated cardiovascular risk in patients with obstructive sleep apnea (OSA). We investigated the relationship between apnea severity and hemostasis factors and effect of continuous positive airway pressure (CPAP) treatment on hemostatic activity. We performed full overnight polysomnography in 44 OSA patients (mean age 47+/-10 years), yielding apnea-hypopnea index (AHI) and mean nighttime oxyhemoglobin saturation (SpO2) as indices of apnea severity. For treatment, subjects were double-blind randomized to 2 weeks of either therapeutic CPAP (n = 18), 3 l/min supplemental nocturnal oxygen (n = 16) or placebo-CPAP (<1 cm H2O) (n = 10). Levels of von Willebrand factor antigen (VWF:Ag), soluble tissue factor (sTF), D-dimer, and plasminogen activator inhibitor (PAI)-1 antigen were measured in plasma pre- and posttreatment. Before treatment, PAI-1 was significantly correlated with AHI (r = 0.47, p = 0.001) and mean nighttime SpO2 (r = -0.32, p = 0.035), but these OSA measures were not significantly related with VWF:Ag, sTF, and D-dimer. AHI was a significant predictor of PAI-1 (R2 = 0.219, standardized beta = 0.47, p = 0.001), independent of mean nighttime SpO2, body mass index (BMI), and age. A weak time-by-treatment interaction for PAI-1 was observed (p = 0.041), even after adjusting for age, BMI, pre-treatment AHI, and mean SpO2 (p = 0.046). Post hoc analyses suggested that only CPAP treatment was associated with a decrease in PAI-1 (p = 0.039); there were no changes in VWF:Ag, sTF, and D-dimer associated with treatment with placebo-CPAP or with nocturnal oxygen. Apnea severity may be associated with impairment in the fibrinolytic capacity. To the extent that our sample size was limited, the observation that CPAP treatment led to a decrease in PAI-1 in OSA must be regarded as tentative.

TAFI and PAI-1 levels in human sepsis

BACKGROUND: Plasminogen activator inhibitor type-1 (PAI-1) is considered to be the main inhibitor of fibrinolysis in sepsis. However, the contribution of TAFI to the inhibition of fibrinolysis in sepsis is currently unknown. METHODS: TAFI antigen and PAI-1 levels were measured in severe sepsis (n = 32) and septic shock (n = 8) patients. In addition, TAFI antigen levels had been determined in 151 controls. RESULTS: Septic patients had significantly (p < 0.0001) decreased TAFI levels (median: 78.9% [range: 32.4-172.6]) as compared to controls (108.1% [35.9-255.4]). TAFI levels were equal in septic shock and severe sepsis (68.9% [32.4-172.6] vs. 82.5% [32.7-144.9], p = 0.987) as well as in survivors and non-survivors (87.1% [32.7-172.6] vs. 65.8% [32.4-129.5], p = 0.166). PAI-1 levels were significantly (705.5 ng/ml [131-5788]) higher in septic shock as in severe sepsis patients (316.5 ng/ml [53-1311], p = 0.016) and were equal in survivors and non-survivors (342 ng/ml [53-1311] vs. 413 ng/ml [55-5788], p = 0.231). TAT/PAP ratio (R((TAT/PAP))) reflecting the dysbalance between coagulation and fibrinolysis was calculated. R((TAT/PAP)) significantly increased with fatality and was significantly dependent on PAI-1, but not on TAFI. PAI-1 levels (570.5 ng/ml [135-5788]) and R((TAT/PAP)) (1.6 [0.3-6.1]) were significantly (p = 0.008 and p = 0.047) higher in patients with overt DIC as compared to patients without overt DIC (310 ng/ml [53-1128] and 0.6 [0.1-4.3]), whereas no difference was found for TAFI levels (68.9% [32.7-133.2] vs. 86.4% [32.4-172.6], p = 0.325). CONCLUSIONS: Although inhibition in sepsis is mediated by both, PAI-1 might be involved early in the sepsis process, whereas TAFI might be responsible for ongoing fibrinolysis inhibition in later stages of sepsis.

Early recurrent ischemic stroke in stroke patients undergoing intravenous thrombolysis

BACKGROUND: We assessed the incidence of early recurrent ischemic stroke in stroke patients treated with intravenous tissue-type plasminogen activator (tPA) and the temporal pattern of its occurrence compared with symptomatic intracranial hemorrhage (ICH). METHODS AND RESULTS: Prospectively collected, population-based data for 341 consecutive acute stroke patients (62% men; mean age, 66 years) treated with tPA according to the National Institute of Neurological Disorders and Stroke study protocol at 8 medical centers in Switzerland (3 academic and 5 community) between January 2001 and November 2004 were retrospectively analyzed. The primary outcome measure was neurological deterioration > or = 4 points on the National Institutes of Health Stroke Scale occurring within 24 hours of tPA treatment and caused either by recurrent ischemic stroke (defined as the occurrence of new neurological symptoms suggesting involvement of initially unaffected vascular territories and evidence of corresponding ischemic lesions on cranial computed tomography scans, in the absence of ICH) or by ICH. Early recurrent ischemic stroke was diagnosed in 2 patients (0.59%; 95% confidence interval, 0.07% to 2.10%) and symptomatic ICH in 15 patients (4.40%; 95% confidence interval, 2.48% to 7.15%). Both recurrent ischemic strokes occurred during thrombolysis, whereas symptomatic ICHs occurred 2 to 22 hours after termination of tPA infusion. CONCLUSIONS: Recurrent ischemic stroke is a rare cause of early neurological deterioration in acute stroke patients undergoing intravenous thrombolysis, with a different temporal pattern compared with that of symptomatic ICH.

International Society on Thrombosis and Haemostasis score for overt disseminated intravascular coagulation predicts organ dysfunction and fatality in sepsis patients

We evaluated the score for disseminated intravascular coagulation (DIC) recently published by the International Society for Thrombosis and Haemostasis (ISTH) in a well-defined series of sepsis patients. Thirty-two patients suffering from severe sepsis and eight patients with septic shock were evaluated following the ISTH DIC score. Fibrin monomer and D-dimer were chosen as fibrin-related markers (FRM), respectively. DIC scores for nonsurvivors (n = 13) as well as for septic shock patients were higher (P < 0.04) compared with survivors and patients with severe sepsis, respectively. Using fibrin monomer and D-dimer, 30 and 25% of patients suffered from overt DIC. Overt DIC was associated with significantly elevated thrombin-antithrombin complexes and plasminogen activator inhibitor type-1 levels as well as with significantly lower factor VII clotting activity. Patients with overt DIC had a significantly higher risk of death and of developing septic shock. Since more than 95% of the sepsis patients had elevated FRM, the DIC score was strongly dependent on prolongation of the prothrombin time and platelet counts. The ISTH DIC score is useful to identify patients with coagulation activation, predicting fatality and disease severity. It mainly depends on the prolongation of the prothrombin time and platelet counts.

Platelet collagen receptors

Collagens are important platelet activators in the vascular subendothelium and vessel wall. Since the regulation of platelet activation is a key step in distinguishing normal haemostasis from pathological thrombosis, collagen interactions with platelets are important targets for pharmacological control. Platelets have two major receptors for collagens, the integrin alpha2beta1, with a major role in adhesion and platelet anchoring and the Ig superfamily member, GPVI, principally responsible for signalling and platelet activation. In addition, GPIb-V-IX, can be considered as an indirect collagen receptor acting via von Willebrand factor as bridging molecule and is essential for platelet interactions with collagen at high shear rates. There is some evidence for additional receptors, which may regulate the response to individual collagen types. This review discusses how these receptors work separately with specific agonists and proposes possible mechanisms for how they work together to regulate platelet activation by collagen, which remains controversial and poorly understood.

Identification of polymorphisms in the human 11beta-hydroxysteroid dehydrogenase type 2 gene promoter: functional characterization and relevance for salt sensitivity

Reduced activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a role in essential hypertension and the sensitivity of blood pressure to dietary salt. Nonconservative mutations in the coding region are extremely rare and do not explain the variable 11beta-HSD2 activity. We focused therefore on the 5'-regulatory region and identified and characterized the first promoter polymorphisms. Transfections of variants G-209A and G-126A into SW620 cells reduced promoter activity and affinity for activators nuclear factor 1 (NF1) and Sp1. Chromatin immunoprecipitation revealed Sp1, NF1, and glucocorticoid receptor (GR) binding to the HSD11B2 promoter. Dexamethasone induced expression of mRNA and activity of HSD11B2. GR and/or NF1 overexpression increased endogenous HSD11B2 mRNA and activity. GR complexes cooperated with NF1 to activate HSD11B2, an effect diminished in the presence of the G-209A variant. When compared to salt-resistant subjects (96), salt-sensitive volunteers (54) more frequently had the G-209A variant, higher occurrence of alleles A4/A7 of polymorphic microsatellite marker, and higher urinary ratios of cortisol to cortisone metabolites. First, we conclude that the mechanism of glucocorticoid-induced HSD11B2 expression is mainly mediated by cooperation between GR and NF1 on the HSD11B2 promoter and, second, that the newly identified promoter variants reduce activity and cooperation of cognate transcription factors, resulting in diminished HSD11B2 transcription, an effect favoring salt sensitivity.

Success rate and efficiency of activator treatment

In a retrospective multicentre study, the success rate and efficiency of activator treatment were analysed. All patients from two University clinics (Giessen, Germany and Berne, Switzerland) that fulfilled the selection criteria (Class II division 1 malocclusion, activator treatment, no aplasia, no extraction of permanent teeth, no syndromes, no previous orthodontic treatment except transverse maxillary expansion, full available records) were included in the study. The subject material amounted to 222 patients with a mean age of 10.6 years. Patient records, lateral head films, and dental casts were evaluated. Treatment was classified as successful if the molar relationship improved by at least half to three-fourths cusp width depending on whether or not the leeway space was used during treatment. Group comparisons were carried out using Wilcoxon two-sample and Kruskal-Wallis tests. For discrete data, chi-square analysis was used and Fisher's exact test when the sample size was small. Stepwise logistic regression was also employed. The success rate was 64 per cent in Giessen and 66 per cent in Berne. The only factor that significantly (P < 0.001) influenced treatment success was the level of co-operation. In approximately 27 per cent of the patients at both centres, the post-treatment occlusion was an 'ideal' Class I. In an additional 38 per cent of the patients, marked improvements in occlusal relationships were found. In subjects with Class II division 1 malocclusions, in which orthodontic treatment is performed by means of activators, a marked improvement of the Class II dental arch relationships can be expected in approximately 65 per cent of subjects. Activator treatment is more efficient in the late than in the early mixed dentition.

Comparison of intraarterial and intravenous thrombolysis for ischemic stroke with hyperdense middle cerebral artery sign

BACKGROUND AND PURPOSE: It is unclear whether intraarterial (IAT) or intravenous (IVT) thrombolysis is more effective for ischemic stroke with hyperdense middle cerebral artery sign (HMCAS) on computed tomography (CT). The aim of this study was to compare IAT and IVT in stroke patients with HMCAS. METHODS: Comparison of data from 2 stroke units with similar management of stroke associated with HMCAS, except that 1 unit performed IAT with urokinase and the other IVT with plasminogen activator. Time to treatment was up to 6 hours for IAT and up to 3 hours for IVT. Outcome was measured by mortality and the modified Rankin Scale (mRS), dichotomized at 3 months into favorable (mRS 0 to 2) and unfavorable (mRS 3 to 6). RESULTS: One hundred twelve patients exhibited a HMCAS, 55 of 268 patients treated with IAT and 57 of 249 patients who underwent IVT. Stroke severity at baseline and patient age were similar in both groups. Mean time to treatment was longer in the IAT group (244+/-63 minutes) than in the IVT group (156+/-21 minutes; P=0.0001). However, favorable outcome was more frequent after IAT (n=29, 53%) than after IVT (n=13, 23%; P=0.001), and mortality was lower after IAT (n=4, 7%) than after IVT (n=13, 23%; P=0.022). After multiple regression analysis IAT was associated with a more favorable outcome than IVT (P=0.003) but similar mortality (P=0.192). CONCLUSIONS: In this observational study intraarterial thrombolysis was more beneficial than IVT in the specific group of stroke patients presenting with HMCAS on CT, even though IAT was started later. Our results indicate that a randomized trial comparing both thrombolytic treatments in patients with middle cerebral artery occlusion is warranted.