Learning Curve for Flow Diversion of Posterior Circulation Aneurysms: A Long-Term International Multicenter Cohort Study.

BACKGROUND AND PURPOSE: Flow diversion has gradually become a standard treatment for intracranial aneurysms of the anterior circulation. Recently, the off-label use of the flow diverters to treat posterior circulation aneurysms has also increased despite initial concerns of rupture and the suboptimal results. This study aimed to explore the change in complication rates and treatment outcomes across time for posterior circulation aneurysms treated using flow diversion and to further evaluate the mechanisms and variables that could potentially explain the change and outcomes. MATERIALS AND METHODS: A retrospective review using a standardized data set at multiple international academic institutions was performed to identify patients with ruptured and unruptured posterior circulation aneurysms treated with flow diversion during a decade spanning January 2011 to January 2020. This period was then categorized into 4 intervals. RESULTS: A total of 378 procedures were performed during the study period. Across time, there was an increasing tendency to treat more vertebral artery and fewer large vertebrobasilar aneurysms (P = .05). Moreover, interventionalists have been increasingly using fewer overlapping flow diverters per aneurysm (P = .07). There was a trend toward a decrease in the rate of thromboembolic complications from 15.8% in 2011-13 to 8.9% in 2018-19 (P = .34). CONCLUSIONS: This multicenter experience revealed a trend toward treating fewer basilar aneurysms, smaller aneurysms, and increased usage of a single flow diverter, leading to a decrease in the rate of thromboembolic and hemorrhagic complications.

PO-13 - Production of a functional thrombopoietin by a human ovarian cancer cell line.

INTRODUCTION: Hemostatic abnormalities are frequently noticed in patients with malignant diseases. These complications include platelets disorders. The role of platelets in cancer extends beyond thrombocytosis and thrombosis, and also platelets promote cancer growth and metastatic dissemination. In the physiology, platelet production is regulated by thrombopoietin, which is mainly secreted by the liver. We, previously, reported that thrombopoietin could be secreted by the ovarian adenocarcinoma cell line, OVCAR-3. AIM: Our main purpose is to analyze the gene expression of thrombopoietin in ovarian cancer cells and to assess its functionality. MATERIALS AND METHODS: The thrombopoietin gene expression in ascitic cells from patients with ovarian carcinomatosis, as well as, in three cancer cell lines, including OVCAR-3 cells, performed using reverse transcription PCR, real-time PCR and gene sequencing, Normal human ovary and liver tissues are used as controls. The functionality of thrombopoietin on the basis of the viability of a thrombopoietin-dependent cell line (Ba/F3) using a co-culture method. RESULTS: Similarly to liver and ovary tissues, all cancer cells lines express the three TPO-1 (full length TPO), TPO-2 (12bp deletion) and TPO-3 (116pb deletion) variants. By flow cytometry, we show that thrombopoietin production by OVCAR-3 could be increased when cells are stimulated by activated protein C. Lastly, Our results confirm that activated protein C may act, in a paracrine fashion, to boost thrombopoietin production. CONCLUSIONS: We report, for the first time, that thrombopoietin secreted by ovarian cancer cells is functional. Hence, thrombopoietin produced by tumor cells may have a direct effect on thrombocytosis/thrombosis occurrence in patients with ovarian cancer.

PO-23 - Expression of heparanase in cancer as biomarker of malignancies: overexpression in an aggressive, poor survival gastric cancer "gastric signet ring cell carcinoma" compared with that of other gastric cancers.

INTRODUCTION: Gastric signet ring cell carcinoma (GSRC) is a distinct entity among of other gastric cancer. With unknown etiopathology, their incidence is increasing and it presents a low sensitivity to chemotherapy. AIM: Here, we studied the expression of the heparanase (HPA) in cancer tissues from GSRC patients and several cancer cell lines. HPA is an endo-ß-D-glucuronidase, capable of cleaving the lateral chains of heparan sulfate on cell surfaces and the extracellular matrix at acidic pH. Apart from its well-characterized enzyme activity, HPA also has independent enzymatic functions, such as up-regulation of vascular endothelial growth factor (VEGF) -A and VEGF-C and activation of intracellular signaling pathways involved in, survival, migration and proliferation of tumor cells. It can also induce an hypercoagulability by a non-enzymatic manner. MATERIALS AND METHODS: HPA was tested in several cancer cell lines from ovaries (OVCAR-3, SKOV-3), breast (MDAMB231, MCF7) colon (LS-174), lung (A549), uterus (HELA) and gastric (adenocarcinoma (AGS) and GSRC (KATO-III) using several techniques such as RT-PCR, Q-PCR, immunocytochemistry, flow cytometry and degradation of Fondaparinux at pH 5, evaluated by its anti Xa activity evaluated by Factor Xa amidolytic activity. The amount of HPA mRNA in the biopsy simples of gastric adenocarcinoma (n=10) and GSRC (n=11) in tumors and their environment were analyzed. RESULTS: HPA gene is expressed in all cancer cell lines, but its level varies depending on the tumor cell line. In biopsies of gastric cancer, the HPA gene is more expressed in the tumor regions (p=0.0002) and tumor environment (p=0.015) in GSRC than in gastric adenocarcinoma. B) The activity of HPA, evaluated by degradation of Fondaparinux at pH 5, 1) in the supernatants of 10(6) cancer cells: the residual activity of Fondaparinux after 2 hours incubation at 37°C with OVCAR-3 supernatant was of 70% of control value, and of 80% with KATO-III cell supernatants. 2) in patient's plasmas, this technique cannot be used because the site of degradation of fondaparinux by heparanase is masked by AT present in plasma. CONCLUSIONS: Heparanase was found in in many cancer cell lines and its level depends on origin of tumor cells and on its aggressivity. Taking into account the pro-metastatic functions, proangiogenic and procoagulant activity of HPA and its overexpression in gastric signet ring cell carcinoma of poor prognosis and its cell line, HPA can be considered as a biomarker of malignancy and as a therapeutic target in GSRC patients.

PO-47 - Microparticles derived from ovarian cancer cell line contained genomic and biologically active proteins, including tissue factor involved in coagulation.

INTRODUCTION: The microparticles (MPs) are sized vesicles of less than 1 µm, from different cell types upon activation or subsequent to apoptosis. They are involved in the thrombotic process, particularly in cancer. The role of MPs in ovarian cancer and their involvement in thrombosis being poorly understood. AIM: The aim of this study was to identify in vitro the generation of MPs by an human ovarian adenocarcinoma cell line (OVCAR-3). MATERIALS AND METHODS: OVCAR-3 cells were cultured in three conditions [without stimulation, with protein C (PC), and with activated protein C (APC)]. Then, the MPs present in the supernatant, were isolated by ultracentrifugation and were analyzed for their shape and properties by flow cytometry, electron microscopy, cryofracture analysis, DNA and RNA, and proteomic analysis. The level of tissue factor (TF) on MP was evaluated by TF-induced shortening of Ca(2+) plasma coagulation time. RESULTS: Our results demonstrated that 1) 92% of MPs derived from OVCAR-3 were less than 100 nm. 2) As tested by flow cytometry, the MPs contained b2 microglobulin, annexin, DNA fragments and TF that induces a shortening of Ca(2+) -induced plasma coagulation time. When OVCAR-3 were cultured for 18H with PCA, MPs were generated in greater amount than those generated by OVCAR-3 in its absence and their level of TF was increased of 20%. Curiously, in contrast with intact OVCAR-3 cells, the endothelial protein C receptor (EPCR) was not detected in MPs 3) Proteomic analysis show that the MPs contain proteins involved in cancer progression such as mucins (5A and 5B), keratin type-1, actin, annexin (A1, A2, A4), CD44, glypican, heat shock (70kDa and HS90a) proteins, Agrin associated with heparan sulfate proteoglycan abundant in the tumor-specific basement membrane, Ephrin type A receptor, coronin-1C, catenin α, integrin ß-1 and also p-selectin responsible of platelet activation. They also express several DNA associated proteins includingtranscription factors, various polymerases, nucleases, and histones involved in chromosome packaging and transcription in the cell nucleus. CONCLUSIONS: MPs derived from OVCAR-3 have an apoptotic character. They expressed several biologically active proteins, DNA and their associated proteins. Despite the absence of EPCR expression on MPs that was expressed on intact OVCAR-3 cells, they expressed procoagulant TF activity already found on intact ovarian cancer cells. This activity is greater extent in the presence of APC.

Analysis of endothelial protein C receptor functionality on living cells'.

Activated protein C (APC) is a major control system of blood coagulation. APC prevents coagulation pathway by degrading Va and VIIIa plasma's coagulation factors. Protein C activation requires its binding to specific endothelial cell receptor (EPCR). APC binding to EPCR also activates a wide range of defense mechanisms (anti-inflammatory, antiapoptosis...). EPCR expression by cells can be detected by various methods, including immunoanalysis and molecular biology. However, no assays evaluate its functionality. A method, inspired of a standard fibrinoformation time assay, was developed to estimate EPCR ability to bind APC on living cell surface in vitro. Endothelial cells were incubated with APC and fibrinoformation on cells was followed by spectrophotometry (plasma absorbance increases with fibrin polymerization). Membrane-bound EPCR retain APC, thus prolonging fibrinoformation time in a dose-dependent manner. Control was realized with EPCR-negative cells. This new method can be used on any cell type to study the expression of other coagulation receptors.

Dose effect relationship of reviparin in chronic hemodialysis: a crossover study versus nadroparin.

Low molecular weight heparins (LMWHs) are used for prevention of clotting in the dialysis circuit. The aim of this trial was to define the optimal dose of a new LMWH and to test the efficiency of a single dose at the start of the session. Fifteen patients were treated according to a double blind and crossover design during 4 blocks of 5 consecutive reviparin doses assigned randomly as 50, 60, 70, 85, and 100 IU anti-Xa/kg. Assessment was carried out on screening of fibrin rings or clots in the arterial and venous air traps and on visual detection of fiber in the dialyzer after rinsing. These clinical results were compared to plasmatic anti-Xa activity and thrombin-antithrombin (TAT) complex generation. A standard dose of 70 IU anti-Xa/kg of nadroparin was used as the control. After a bolus of 50 to 100 IU anti-Xa/kg, the occurrence of fibrin rings and clots in the air traps was dependent on three factors: dose of LMWH, time of the session, and patient status. A bolus of 85 IU anti-Xa/kg of reviparin was effective and safe for sessions of 4 h. For this dose, plasmatic anti-Xa activity was 0.96 +/- 0.28 IU/ml at Hour 2 and 0.82 +/- 0.22 IU/ml at Hour 4. TAT complexes are good markers of the activation of the coagulation. They did not increase during a 4 h session after a reviparin bolus of 100 IU/kg. For the same LMWH dose, the trial shows a great variability of the clinical effect and anti-Xa activities from one patient to another. A single dose of 85 IU anti-Xa/kg of reviparin can be used at the start of the dialysis session as a loading dose. We advise adapting the dose during the subsequent sessions according to the appearance of the blood circuit. The benefit of monitoring anti-Xa activity and TAT complexes could be tested in a further trial.

Mineralocorticoid hormone signaling regulates the 'epithelial sodium channel' in fibroblasts from human cornea.

We investigated the regulation of sodium absorption by steroid hormones in embryologically diverse cells from the human eye. A cell extract from human corneal fibroblasts was positive for both the epithelial sodium channel (ENaC) and the mineralocorticoid receptor (MCR) as 82- to 85-kD and 102-kD bands, respectively, by the Western blot technique. In fluorescent, confocal and electron microscopy, the MCR was revealed as a nucleocytoplasmic protein, whereas the ENaC was almost exclusively membrane bound; both appeared aligned along actin filaments of corneal keratocytes, and both were widely colocalized in various cell types of human cornea in situ. Following reverse transcription and amplification of total RNA isolated from corneal fibroblasts, the ENaC and MCR genes in the PCR product were evident as predicted bands of 520 and 843 bp, respectively, whose sequence exhibited 100% identity with those from known human sources. The multiplication of corneal fibroblasts was influenced by both the MCR-specific antagonist RU 26752 and the natural hormone aldosterone, and these steroids also stimulated protein phosphorylation. In quantitative PCR, both the basal and aldosterone-induced levels of ENaC were diminished by the MCR-specific antagonist ZK 91587. Consequently, the ocular sodium channel appears to be regulated by steroid signalling in cells of diverse embryological origins, contrary to the existing notions where (a) this process would be limited exclusively to the epithelial cells and (b) ocular sodium transport would be regulated via the Na(+)-K(+)-ATPase in the basolateral membrane.

Fibrinogen Alès: a homozygous case of dysfibrinogenemia (gamma-Asp(330)-->Val) characterized by a defective fibrin polymerization site "a".

Congenital homozygous dysfibrinogenemia was diagnosed in a man with a history of 2 thrombotic strokes before age 30. His hemostatic profile was characterized by a dramatically prolonged plasma thrombin clotting time, and no clotting was observed with reptilase. Complete clotting of the abnormal fibrinogen occurred after a prolonged incubation of plasma with thrombin. The release of fibrinopeptides A and B by thrombin and of fibrinopeptide A by reptilase were both normal. Thrombin-induced fibrin polymerization was impaired, and no polymerization occurred with reptilase. The polymerization defect was characterized by a defective site "a," resulting in an absence of interaction between sites A and a, indicated by the lack of fragment D(1) (or fibrinogen) binding to normal fibrin monomers depleted in fibrinopeptide A only (Des-AA fm). By SDS-PAGE, the defect was detected on the gamma-chain and in its fragment D(1). The molecular defect determined by analysis of genomic DNA showed a single base change (A-->T) in exon VIII of the gamma-chain. The resulting change in the amino acid structure is gamma 330 aspartic acid (GAT) --> valine (GTT). It is concluded that the residue gamma-Asp(330) is essential for the normal functioning of the polymerization site a on the fibrinogen gamma-chain.

Thrombotic dysfibrinogenemia. Fibrinogen "Caracas V" relation between very tight fibrin network and defective clot degradability.

Fibrinogen Caracas V is a thrombotic dysfibrinogenemia with an Aalpha 532 Ser-->Cys mutation characterized by a tight fibrin network formed of thin fibers responsible for a less porous clot than a normal one. In the present work, fibrinogen Caracas V is further characterized in order to understand the relationship between the structural defect and thrombophilia. This thrombotic disorder has been attributed to a tight fibrin network responsible for a decreased permeation of flow through the clot, leading to defective thrombus lysis due to a diminished availability of fibrinolytic enzymes to the inner fibrin surface. Correction of clot structure anomaly, by addition of dextran 40 to fibrinogen before clotting, induces an improvement in fibrin degradation that was attributed to an increase in porosity. The pulmonary embolism observed in this family has been related to an hyper rigidity of the clot, an anomaly that is also corrected by dextran. Furthermore, this abnormal fibrinogen binds more albumin than does normal fibrinogen, a phenomenon attributed to the mutation of serine in Aalpha-532 by cysteine. Therefore, this fibrinogen shows a striking similarity to the fibrinogen Dusart, allowing us to confirm that the alphaC-terminal part of fibrinogen plays an important role in fibrin structure, and to conclude that the anomaly of fibrin network observed in fibrinogen Caracas V is responsible for a deficient thrombus lysis.

Demonstration of the mineralocorticoid hormone receptor and action in human leukemic cell lines.

We studied the expression of the mineralocorticoid receptor (MCR), and of the amiloride-sensitive sodium channel (ASSC) regulated by the MCR, in human leukemic cell lines. Cell extracts from TF1 (proerythroblastic), HEL (human erythroblastic leukemia) and U937 (myeloblastic) cell line were positive for the ASSC, as a 82 kDa band in Western blots developed with the aid of a polyclonal antibody raised against the peptide QGLGKGDKREEQGL, corresponding to the region 44-58 of the alpha subunit of the epithelial sodium channel (ENaC) cloned from rat colon, linked to KLH. The polyclonal antibody against the MCR revealed a single band of about 102 kDa in extracts from HEL and TF1 cells. The immunofluorescent labelling of the MCR in all cell lines showed a nucleocytoplasmic localization of the receptor but the ASSC was exclusively membrane-bound and these results were confirmed by confocal microscopy. The expression of the MCR in the HEL cells was evident as a predicted band of 843 bp (234 amino acids) in electrophoresis of the PCR product obtained after total RNA had been reverse transcribed and then amplified using the primers 5'-AGGCTACCACAGTCTCCCTG-3' and 5'-GCAGTGTAAAATCTCCAGTC-3' (sense and antisense, respectively). The ENaC was similarly evident with the aid of the primers 5'-CTGCCmATG GATGATGGT-3' (sense) and 5'-GTTCAGCTCGAAGAAGA-3' (antisense) as a predicted band of 520 bp. In both cases, 100% identity was observed between the sequences of the PCR products compared to those from known human sources. The multiplication of the HEL cells was influenced by antagonists (RU 26752, ZK 91587) targeted for specificity to the MCR and this was selectively reversed by the natural hormone aldosterone. These steroids also provoked chromatin condensation in the HEL population. These permit new and novel possibilities to understand the pathobiology of human leukemia and to delineate sodium-water homeostasis in nonepithelial cells.

Immunochemical analysis of the sodium channel in rodent and human eye.

The presence of the amiloride-sensitive sodium channel (ASSC) in ocular tissues was studied with the aid of a polyclonal antiserum raised against the 14 amino acid peptide QGLGKGDKREEQGL. This sequence corresponds to the region 44-58 of the alpha subunit of the channel, termed ENaC, cloned from rat colon. The antibody titers, measured by the ELISA technique, rose to 1∶2560 4 weeks after immunization, and this bleed was used in all subsequent experiments. Immunoblotting with the polyclonal anti-alphaENaC serum, revealed a major band of 82-86 kDa in extracts prepared from whole bovine or rat retina; a minor component of 92 kDa in the extract from bovine ciliary body may represent a glycosylated species. Immunohistochemistry, using the alphaENaC-specific antiserum, revealed strong fluorescence in specific areas of the rat and human eye. Pronounced labelling was observed in the epithelial cell layer of the retina, the lens, as well as both the pigmented and the nonpigmented epithelium of the ciliary body and the iris. All of the cell layers (epithelium, endothelium and fibroblasts) in the cornea, the blood vessels in the iris, and iris epithelium, were also strongly immunopositive. The somatic body of the photoreceptor cells (cones and rods) in the inner and outer segments could be traced to forming a synapse in both the internal and external portions of the internal nuclear layer. The bipolar cells and ganglia in the neuronal compartment also exhibited occasional immunofluorescence. The method of fixation and the source of the tissue were important parameters for the immunochemical localization of the ENaC. The resolution was very poor when rat eye was fixed in Bouin's solution but this method was satisfactory for human tissues. For rat eye, optimum resolution was obtained with AMeX fixation. This widespread distribution of the ENaC generally colocalizes with the previously observed immunopositivity for the mineralocorticoid receptor such that steroid hormone-mediated ion regulation would appear to add a new parameter to the functional expression of ocular tissues.

Thrombus generation after adenovirus-mediated gene transfer into atherosclerotic arteries.

Thrombosis represents a major issue during arterial local delivery. We evaluated the occurrence of thrombosis after adenovirus (Ad)-mediated gene transfer into normal and atherosclerotic arteries. A replication-deficient Ad vector expressing the beta-galactosidase reporter gene (Ad.RSV betagal; 4 x 10(9) PFU) was injected into normal and atherosclerotic arteries (n = 11 in both groups). The contralateral artery received either an Ad vector carrying no transgene (Ad.MLPnull) (n = 7 in both groups, 4 x 10(9) PFU) or vehicle buffer (n = 4 in normal group, n = 8 in atherosclerotic group). Animals were sacrificed 3 days following gene transfer for thrombus detection and assessment of beta-galactosidase activity. Thrombus was absent in normal arteries and in atherosclerotic arteries injected with vehicle buffer only. In contrast, nonocclusive thrombus was present in atherosclerotic arteries injected with either Ad.RSV betagal (5 of 11) or Ad.MLPnull (3 of 7). Beta-galactosidase activity was predominantly found in the endothelial layer of the transfected arteries. Gene transfer and expression occurred despite the presence of the thrombus (4 of 5), and its efficiency did not significantly differ regardless of the thrombus. We conclude that thrombus frequently occurred in atherosclerotic arteries after Ad-mediated gene transfer. Further studies are warranted to identify the mechanisms of thrombus generation after Ad-mediated gene transfer into atherosclerotic arteries.

Mineralocorticoid hormone receptor and the epithelial sodium channel in a human leukemic cell line.

A Cell extract from the HEL (human erythroblastic leukemia) cell line was positive for both the epithelial sodium channel (ENaC) and the mineralocorticoid receptor (MCR) as glycosylated 82-84 kDa bands, and a single 102 kDa band, respectively, in Western blots using polyclonal antibodies raised against these proteins. The immunofluorescent labeling of the MCR in all cell lines showed a nucleocytoplasmic localization of the receptor whereas the ENaC was exclusively membrane-bound. These results were confirmed by confocal microscopy. The expression of the MCR in HEL cells was evident as a predicted band of 843 bp (234 amino acids) after total RNA from HEL cells had been reverse transcribed and then amplified by PCR; the ENaC was similarly evident as a predicted band of 520 bp. In both cases, near 100% identity was observed between the deduced amino acid sequences of the PCR products and those from known human sources. The multiplication of HEL cells was influenced by antagonists (RU 26752, ZK 91587) targeted for specificity to the MCR and this was reversed by the natural hormone aldosterone. These steroids also provoked chromatin condensation in the HEL population.

Defective cell migration in an ovarian cancer cell line is associated with impaired urokinase-induced tyrosine phosphorylation.

The urokinase receptor (u-PAR), a protein anchored to cell membrane by a glycosyl phosphatidylinositol, plays a central role in cancer cell invasion and metastasis by binding urokinase plasminogen activator (u-PA), thereby facilitating plasminogen activation. Plasmin can promote cell migration either directly or by activating metalloproteinases that degrade some of the components of the extra cellular matrix. However, the IGR-OV1-Adria cell line contains the u-PAR but does not migrate even in the presence of exogenous u-PA, although the parental IGR-OV1 cell line migrates normally in the presence of u-PA. We therefore investigated the role of cell signalling for u-PA induced cell locomotion. We show that cell migration induced by u-PA-u-PAR complex is always associated with tyrosine kinase activation for the following reasons: (1) the blockade of the u-PAR by a chimeric molecule (albumin-ATF) inhibits not only the u-PA-induced cell migration, but also the signalling in IGR-OV1 line; (2) the binding of u-PA to u-PAR on non-migrating IGR-OV1-Adria cells was not associated with tyrosine kinase activation; (3) the inhibition of tyrosine kinase also blocked cell migration of IGR-OV1. Therefore tyrosine kinase activation seems to be essential for the u-PA-induced cell locomotion possibly by the formation of a complex u-PAR-u-PA with a protein whose transmembrane domain can ensure cell signalling. Thus, IGR-OV1 and IGR-OV1-Adria cell lines represent a good model for the analysis of the mechanism of u-PA-u-PAR-induced cell locomotion.

Down-regulation of fibrinogen biosynthesis by IL-4, IL-10 and IL-13.

High levels of fibrinogen are recognized as an important vascular risk factor; however, it is not known if the increase of plasma fibrinogen is directly responsible for this risk, or is only a marker of vascular inflammation. To support this second hypothesis, Oncostatin M (OSM) is a potent stimulator of fibrinogen biosynthesis and induces smooth muscle cell proliferation. In the same way, we analysed whether interleukin-4 (IL-4), interleukin-10 (IL-10) or interleukin-13 (IL-13), which protect vessel walls from monocytes injuries leading to atherosclerosis, could influence fibrinogen biosynthesis. The two levels of regulation of fibrinogen biosynthesis were tested: firstly, the direct effect of these cytokines on fibrinogen production by the hepatoma cell line Hep G2, and secondly their effect on the secretion of hepatocyte stimulating factor (HSF) activity in the supernatant of lipopolysaccharide (LPS)-activated monocytes. IL-4 and IL-13 added to Hep G2 cells down-regulated both the increase of fibrinogen secretion induced by IL-6 and fibrinogen mRNA levels, this effect being more pronounced when Hep G2 were preincubated with the two cytokines before IL-6 addition. The effect of IL-10 was evidenced only on mRNA expression. IL-10 and IL-13 dose-dependently decrease HSF activity secreted by LPS-activated monocytes, whereas IL-4 had no effect. However, the three cytokines decreased HSF activity when monocytes were incubated with the cytokines before LPS activation. The effects of these cytokines on HSF activity are related to variations of IL-6 and OSM secretion. Our data strengthen the hypothesis that the fibrinogen level is a marker of vascular disease, since cytokines which have a protective vascular effect down-regulate fibrinogen production.

Expression of elastase and fibrin in venous leg ulcer biopsies: a pilot study of pentoxifylline versus placebo.

The pathogenesis of venous leg ulcers is based on the leakage of fibrinogen leading to a pericapillary fibrin cuff and plugging of capillaries by white blood cells. On the basis of a previous work, we had assumed that the key event in the pathogenesis of venous leg ulcers is related to inflammation generated by activated white blood cells that accumulate under unrelieved blood pressure, because in ulcer biopsies we had detected the presence of tumor necrosis factor-alpha (TNF-alpha) in intracapillary monocytes, elastase in the polymorphonuclear leukocytes near the vessels, and a pericapillary undegraded fibrin cuff causing a diffusion barrier to oxygen. This concept was developed because TNF-alpha synthesized by activated monocytes is responsible for many deleterious effects. It has a potent mitogenic effect on fibroblasts, leading to new collagen deposition and angiogenesis, it induces an increase in collagenase production, it acts through upregulation of an intracellular adhesion molecule (ICAM-1), leading to leukocyte sequestration and consequently a release of toxic metabolites by the polymorphonuclear cells, an early step in chronic inflammation, it activates the coagulation pathway via a marked increase in monocyte-associated tissue factor (TF) procoagulant activity, and it inhibits fibrinolysis by promoting the release of PAI-1, contributing to undegraded fibrin deposition. Therefore, we were interested in evaluating, in patients with venous leg ulcers, the effect of pentoxifylline administered at 1,200 mg daily (versus placebo) for 2-months, as this drug induces a decrease in TNF-alpha synthesis and also blocks its activity. This pilot assay was performed in blind. Evolution of several parameters in ulcer biopsies are analyzed: TNF-alpha, intact fibrin, fibrin degradation products, ICAM-1, TF, and elastase. Pentoxifylline administration induced a decrease of local elastase and of fibrin deposit. These results support the hypothesis that accumulation of activated leukocytes is the key event in venous leg ulcers.

The plasminogen-activation system in ovarian tumors.

We studied the plasminogen activation system in tumor tissue by measuring the antigen level of the 2 plasminogen activators, tissue-type (t-PA) and urokinase-type (U-PA) and their inhibitors, plasminogen-activator inhibitors type-I (PAI-I) and type-2 (PAI-2) in the tissue extracts of 43 human benign and malignant ovarian tumors. U-PA levels were significantly higher in malignant than in benign tumors. In addition, U-PA antigen levels were higher in the metastatic tissue of advanced disease (FIGO stage III) than in the primary localized tumor (FIGO stage I/II). Also PAI-I concentrations tended to be higher in malignant than in benign tumors, but this difference was not statistically significant. In contrast, t-PA levels were lower in metastatic than in non-metastatic tumors, whereas PAI-2 levels were unrelated to the stage of ovarian malignancy. These results were integrated in a plasminogen-activation-dependent malignancy index (U-PA x PAI-I/t-PA). This index distinguished the different groups of benign ovarian tumors, localized and metastatic ovarian carcinomas better than U-PA levels. It could be useful as a prognostic indicator in ovarian cancer.

Potent activity of peripheral blood lymphocytes in inducing hepatocyte stimulating factor and urokinase in monocytes.

As fibrinogen is an independent risk factor for arterial thrombosis we were interested in analysing the mechanism controlling fibrinogen biosynthesis. In this work, we showed that incubation of monocytes with lymphocytes increased hepatocyte stimulating factor (HSF) production. Different mechanisms are involved and our results demonstrated that this effect is in part mediated by an increase in interleukin 6 (IL-6) production. However, IL-6 cannot account for the whole effect and other cytokines could be implicated. In addition, we observed a stimulation of urokinase-type plasminogen activator (u-PA) associated with monocytes when these cells were incubated with lymphocytes for 18 h at 37 degrees C. By producing fragment D (fibrinogen degradation product) and D-dimer (fibrin degradation product) this fibrinolytic activity might also contribute to fibrinogen biosynthesis by hepatocytes.