(4-Aminomethyl)phenylguanidine derivatives as nonpeptidic highly selective inhibitors of human urokinase

Increased expression of the serine protease urokinase-type plasminogen activator (uPA) in tumor tissues is highly correlated with tumor cell migration, invasion, proliferation, progression, and metastasis. Thus inhibition of uPA activity represents a promising target for antimetastatic therapy. So far, only the x-ray crystal structure of uPA inactivated by H-Glu-Gly-Arg-chloromethylketone has been reported, thus limited data are available for a rational structure-based design of uPA inhibitors. Taking into account the trypsin-like arginine specificity of uPA, (4-aminomethyl)phenylguanidine was selected as a potential P1 residue and iterative derivatization of its amino group with various hydrophobic residues, and structure–activity relationship-based optimization of the spacer in terms of hydrogen bond acceptor/donor properties led to N-(1-adamantyl)-N′-(4-guanidinobenzyl)urea as a highly selective nonpeptidic uPA inhibitor. The x-ray crystal structure of the uPA B-chain complexed with this inhibitor revealed a surprising binding mode consisting of the expected insertion of the phenylguanidine moiety into the S1 pocket, but with the adamantyl residue protruding toward the hydrophobic S1′ enzyme subsite, thus exposing the ureido group to hydrogen-bonding interactions. Although in this enzyme-bound state the inhibitor is crossing the active site, interactions with the catalytic residues Ser-195 and His-57 are not observed, but their side chains are spatially displaced for steric reasons. Compared with other trypsin-like serine proteases, the S2 and S3/S4 pockets of uPA are reduced in size because of the 99-insertion loop. Therefore, the peculiar binding mode of the new type of uPA inhibitors offers the possibility of exploiting optimized interactions at the S1′/S2′ subsites to further enhance selectivity and potency. Because crystals of the uPA/benzamidine complex allow inhibitor exchange by soaking procedures, the structure-based design of new generations of uPA inhibitors can rely on the assistance of x-ray analysis.

Expression of multilectin receptors and comparative FITC-dextran uptake by human dendritic cells

Dendritic cells (DC) are potent antigen-presenting cells and understanding their mechanisms of antigen uptake is important for loading DC with antigen for immunotherapy. The multilectin receptors, DEC-205 and macrophage mannose receptor (MMR), are potential antigen-uptake receptors; therefore, we examined their expression and FITC-dextran uptake by various human DC preparations. The RT-PCR analysis detected low levels of DEC-205 mRNA in immature blood DC, Langerhans cells (LC) and immature monocyte-derived DC (Mo-DC), Its mRNA expression increased markedly upon activation, indicating that DEC-205 is an activation-associated molecule. In Mo-DC, the expression of cell-surface DEC-205 increased markedly during maturation. In blood DC, however, the cell-surface expression of DEC-205 did not change during activation, suggesting the presence of a large intracellular pool of DEC-205 or post-transcriptional regulation. Immature Mo-DC expressed abundant MMR, but its expression diminished upon maturation. Blood DC and LC did not express detectable levels of the MMR, FITC-dextran uptake by both immature and activated blood DC was 30- to 70-fold less than that of LC, immature Mo-DC and macrophages. In contrast to immature Mo-DC, the FITC-dextran uptake by LC was not inhibited effectively by mannose, an inhibitor for MMR-mediated FITC-dextran uptake. Thus, unlike Mo-DC, blood DC and LC do not use the MMR for carbohydrate-conjugated antigen uptake and alternative receptors may yet be defined on these DC. Therefore, DEC-205 may have a different specificity as an antigen uptake receptor or contribute to an alternative DC function.

Cell surface expression of urokinase receptor in normal mammary epithelial cells and breast cancer cell lines

Background: The urokinase receptor (uPAR) is important in the process of extracellular matrix degradation occurring during cancer cell invasion and metastasis. We wished to quantify uPAR on the surfaces of normal mammary epithelial cells (HMEC) and 6 well-known breast cancer cell lines using flow cytometry. Materials and Methods: Cell surface uPAR was labelled with a monoclonal antibody, and this was detected with a florescent-labelled second antibody and accurately measured using flow cytometry. The measured fluorescent signals of the stained cells were interpolated with those of Quantum Simply Cellular bead standards to determine the number of uPAR sites per cell. Results: The breast cancer cell lines ranged from 13,700 to 50,800 uPAR sites per cell, whilst HMEC cells had only 2,500 sites. Conclusions: This simple and reliable method showed that the expression of cell surface uPAR is higher in the breast cancer cell lines than in the normal mammary cells.

Break-dance: an unusual cause of hammer syndrome.

We report the case of a young break-dancer presenting with hammer syndrome. This syndrome has been correlated with many professional and recreational activities but this is, to our knowledge, the first description of hammer syndrome caused by break-dancing. The etiology, diagnosis and treatment modalities of this rare syndrome are considered.

Plasminogen interaction with Trypanosoma cruzi

The ability of Trypanosoma cruzi to interact with plasminogen, the zimogenic form of the blood serin protease plasmin, was examined. Immunohistochemistry studies revealed that both forms, epimastigotes and metacyclic trypomastigotes, were able to fix plasminogen in a lysine dependant manner. This interaction was corroborated by plasminogen activation studies. Both forms of the parasite enhanced the plasminogen activation by tissue-type plasminogen activator.The maximal enhancements obtained were 15-fold and 3.4-fold with epimastigotes and metacyclic trypomastigotes, respectively, as compared to plasminogen activation in absence of cells. Ligand-blotting analysis of proteins extracted with Triton X-114 from a microsomal fraction of epimastigotes revealed at least five soluble proteins and one hydrophobic protein able to bind plasminogen.

Improving binding affinity and stability of peptide ligands by substituting glycines with D-amino acids.

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer-related protease urokinase-type plasminogen activator (uPA), we observed a glycine residue that has a positive ϕ dihedral angle when bound to the target. We hypothesized that replacing it with a D-amino acid, which favors positive ϕ angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D-serine in the bicyclic peptide indeed improved inhibitory activity (1.75-fold) and stability (fourfold). X-ray-structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ϕ angles are found in many protein-bound peptides. These results suggest that the glycine-to-D-amino acid mutagenesis strategy could be broadly applied.

Characterization of Human Late Outgrowth Endothelial Progenitor-Derived Cells under Various Flow Conditions.

Background: Endothelial progenitor-derived cells (EPC) are a cell therapy tool in peripheral arterial disease and for re-endothelialization of bypasses and stents. Objective: To assess EPC behavior under flow conditions normally found in vivo. Results: EPC were isolated from human cord blood, cultured on compliant tubes and exposed in an in vitro flow system mimicking hemodynamic environments normally found in medium and large arteries. EPC exposed for 24 h to unidirectional (0.3 ± 0.1 or 6 ± 3 dynes/cm(2)) shear stress oriented along flow direction, while those exposed to bidirectional shear stress (0.3 ± 3 dynes/cm(2)) or static conditions had random orientation. Under bidirectional flow, tissue factor (TF) activity and mRNA expression were significantly increased (2.5- and 7.0-fold) compared to static conditions. Under low shear unidirectional flow TF mRNA increased 4.9 ± 0.5-fold. Similar flow-induced increases were observed for TF in mature umbilical vein-derived endothelial cells. Expression of tissue-type plasminogen activator (t-PA), urokinase (u-PA) and monocyte chemotactic protein 1 (MCP1) were reduced by 40-60% in late outgrowth endothelial progenitor-derived cells (LO-EPC) exposed to any flow environment, while MCP1, but not t-PA or u-PA, was decreased in HUVEC. Conclusions: Flow, in particular bidirectional, modifies the hemostatic balance in LO-EPC with increased TF and decreased plasminogen activator expression.

The role of NFI-C liver regeneration and its potential function as a general regulator of regenerative processes

Collectively, research aimed to understand the regeneration of certain tissues has unveiled the existence of common key regulators. Knockout studies of the murine Nuclear Factor I-C (NFI-C) transcription factor revealed a misregulation of growth factor signaling, in particular that of transforming growth factor ß-1 (TGF-ßl), which led to alterations of skin wound healing and the growth of its appendages, suggesting it may be a general regulator of regenerative processes. We sought to investigate this further by determining whether NFI-C played a role in liver regeneration. Liver regeneration following two-thirds removal of the liver by partial hepatectomy (PH) is a well-established regenerative model whereby changes elicited in hepatocytes following injury lead to a rapid, phased proliferation. However, mechanisms controlling the action of liver proliferative factors such as transforming growth factor-ßl (TGF-ß1) and plasminogen activator inhibitor-1 (PAI-1) remain largely unknown. We show that the absence of NFI-C impaired hepatocyte proliferation due to an overexpression of PAI-1 and the subsequent suppression of urokinase plasminogen (uPA) activity and hepatocyte growth factor (HGF) signaling, a potent hepatocyte mitogen. This indicated that NFI-C first acts to promote hepatocyte proliferation at the onset of liver regeneration in wildtype mice. The subsequent transient down regulation of NFI-C, as can be explained by a self- regulatory feedback loop with TGF-ßl, may limit the number of hepatocytes entering the first wave of cell division and/or prevent late initiations of mitosis. Overall, we conclude that NFI-C acts as a regulator of the phased hepatocyte proliferation during liver regeneration. Taken together with NFI-C's actions in other in vivo models of (re)generation, it is plausible that NFI-C may be a general regulator of regenerative processes. - L'ensemble des recherches visant à comprendre la régénération de certains tissus a permis de mettre en évidence l'existence de régulateurs-clés communs. L'étude des souris, dépourvues du gène codant pour le facteur de transcription NFI-C (Nuclear Factor I-C), a montré des dérèglements dans la signalisation de certains facteurs croissance, en particulier du TGF-ßl (transforming growth factor-ßl), ce qui conduit à des altérations de la cicatrisation de la peau et de la croissance des poils et des dents chez ces souris, suggérant que NFI-C pourrait être un régulateur général du processus de régénération. Nous avons cherché à approfondir cette question en déterminant si NFI-C joue un rôle dans la régénération du foie. La régénération du foie, induite par une hépatectomie partielle correspondant à l'ablation des deux-tiers du foie, constitue un modèle de régénération bien établi dans lequel la lésion induite conduit à la prolifération rapide des hépatocytes de façon synchronisée. Cependant, les mécanismes contrôlant l'action de facteurs de prolifération du foie, comme le facteur de croissance TGF-ßl et l'inhibiteur de l'activateur du plasminogène PAI-1 (plasminogen activator inhibitor-1), restent encore très méconnus. Nous avons pu montrer que l'absence de NFI-C affecte la prolifération des hépatocytes, occasionnée par la surexpression de PAI-1 et par la subséquente suppression de l'activité de la protéine uPA (urokinase plasminogen) et de la signalisation du facteur de croissance des hépatocytes HGF (hepatocyte growth factor), un mitogène puissant des hépatocytes. Cela indique que NFI-C agit en premier lieu pour promouvoir la prolifération des hépatocytes au début de la régénération du foie chez les souris de type sauvage. La subséquente baisse transitoire de NFI-C, pouvant s'expliquer par une boucle rétroactive d'autorégulation avec le facteur TGF-ßl, pourrait limiter le nombre d'hépatocytes qui entrent dans la première vague de division cellulaire et/ou inhiber l'initiation de la mitose tardive. L'ensemble de ces résultats nous a permis de conclure que NFI-C agit comme un régulateur de la prolifération des hépatocytes synchrones au cours de la régénération du foie.

Success of thrombolysis as a predictor of outcome in acute thrombosis of popliteal aneurysms.

PURPOSE: Acute limb ischemia after thrombosis of a popliteal aneurysm is a distinct and limb-threatening entity. Preoperative intra-arterial thrombolysis may improve the outcome in this challenging situation. This study retrospectively analyzed a consecutive series of patients treated with preoperative thrombolysis and subsequent revascularization. METHODS: Thirteen patients with acute limb ischemia caused by thrombosis of a popliteal aneurysm underwent catheter-directed intra-arterial thrombolysis with urokinase and subsequent vascular reconstruction. The angiographic and clinical outcome was analyzed and compared with that in the literature. RESULTS: Complete aneurysm thrombosis with absence of runoff was documented in 12 cases. Thrombolysis restored perfusion with patency of the popliteal artery and a one- or two-vessel runoff in 77% of cases (10/13). Early cumulative graft patency and limb salvage rates were 68% and 83%, respectively, with an ankle/brachial index of 0.8 +/- 0.2. Lytic failure followed by attempts at bypass grafting was present in three patients (23%) and resulted in above-knee amputation. Severe rhabdomyolysis and fatal pulmonary embolism were responsible for a 15% early mortality rate. CONCLUSION: Preoperative thrombolysis followed by bypass grafting is a valid treatment option for patients who can withstand an additional period of ischemia that does not require immediate revascularization and intraoperative lysis. Lytic failure identifies patients with a highly compromised runoff who are probably best treated by means of subsequent amputation, without any attempts at bypass grafting.

Susceptibilidad genética para el desarrollo de la sepsis bacteriana grave y choque séptico

Objetivo: presentar el estado del arte de las investigaciones que, hasta el momento, relacionan el polimorfismo genético del paciente con la evolución de la sepsis, como herramienta diagnóstica y un nuevo enfoque terapéutico de esta condición. Los conceptos actuales basados en investigaciones sostienen que el polimorfismo genético del individuo es relevante en la evolución de la enfermedad y en la respuesta efectiva al tratamiento del paciente en estado crítico, en especial con sepsis bacteriana y choque séptico. Materiales y métodos: se revisó literatura indexada que relaciona los factores genéticos con la evolución de algunas enfermedades del paciente en estado crítico. Resultados: las características particulares de la enfermedad estarían influenciadas por el acervo genético del paciente, condicionando en gran medida la respuesta patofisiológica. Se ha evidenciado la susceptibilidad genética de algunos individuos a desarrollar infección; estos individuos con un tratamiento similar no evolucionan de igual forma, desencadenándose una sepsis bacteriana grave y choque séptico. El polimorfismo en los genes que codifican por el factor de necrosis tumoral -α (TNF-α) las interlucinas- 1 (IL-1), IL-6, IL-10, el factor soluble CD-14, los receptores similares a Toll y el inhibidor tipo 1 del activador del plasminógeno estaría asociado con el desarrollo de sepsis grave y choque séptico, en particular las mutaciones TNF-α 308 G/A, PAI-1 4G/4G, IL-6 174 G/C. Conclusiones: el conocimiento de la susceptibilidad genética, los factores de riesgo y el buen funcionamiento del sistema inmune de cada persona ayudan a reducir y compensar las complicaciones de la sepsis bacteriana. Es claro que el tratamiento oportuno individualizado en los pacientes con sepsis se asocia con disminución de la mortalidad y con reducción en el deterioro de la respuesta inflamatoria.

A "Trojan horse" strategy to reverse drug-resistance in brain tumors

Los gliomas malignos representan una de las formas más agresivas de los tumores del sistema nervioso central (SNC). De acuerdo con la clasificación de los tumores cerebrales de la Organización Mundial de la Salud (OMS), los astrocitomas han sido categorizados en cuatro grados, determinados por la patología subyacente. Es así como los gliomas malignos (o de alto grado) incluyen el glioma anaplásico (grado III) así como el glioblastoma multiforme (GBM, grado IV),estos últimos los más agresivos con el peor pronóstico (1). El manejo terapéutico de los tumores del SNC se basa en la cirugía, la radioterapia y la quimioterapia, dependiendo de las características del tumor, el estadio clínico y la edad (2),(3), sin embargo ninguno de los tratamientos estándar es completamente seguro y compatible con una calidad de vida aceptable (3), (4). En general, la quimioterapia es la primera opción en los tumores diseminados, como el glioblastoma invasivo y el meduloblastoma de alto riesgo o con metástasis múltiple, pero el pronóstico en estos pacientes es muy pobre (2),(3). Solamente nuevas terapias dirigidas (2) como las terapias anti-angiogénicas (4); o terapias génicas muestran un beneficio real en grupos limitados de pacientes con defectos moleculares específicos conocidos (4). De este modo, se hace necesario el desarrollo de nuevas terapias farmacológicas para atacar los tumores cerebrales. Frente a las terapias los gliomas malignos son con frecuencia quimioresistentes, y esta resistencia parece depender de al menos dos mecanismos: en primer lugar, la pobre penetración de muchas drogas anticáncer a través de la barrera hematoencefálica (BBB: Blood Brain Barrier), la barrera del fluido sangre-cerebroespinal (BCSFB: Blood-cerebrospinal fluid barrier) y la barrera sangre-tumor (BTB: blood-tumor barrier). Dicha resistencia se debe a la interacción de la droga con varios transportadores o bombas de eflujo de droga ABC (ABC: ATP-binding cassette) que se sobre expresan en las células endoteliales o epiteliales de estas barreras. En segundo lugar, estos transportadores de eflujo de drogas ABC propios de las células tumorales confieren un fenotipo conocido como resistencia a multidrogas (MDR: multidrug resistance), el cual es característico de varios tumores sólidos. Este fenotipo también está presente en los tumores del SNC y su papel en gliomas es objeto de investigación (5). Por consiguiente el suministro de medicamentos a través de la BBB es uno de los problemas vitales en los tratamientos de terapia dirigida. Estudios recientes han demostrado que algunas moléculas pequeñas utilizadas en estas terapias son sustratos de la glicoproteína P (Pgp: P-gycoprotein), así como también de otras bombas de eflujo como las proteínas relacionadas con la resistencia a multidrogas (MRPs: multidrug resistance-related proteins (MRPs) o la proteína relacionada con cáncer de seno (BCRP: breast-cancer resistance related protein)) que no permiten que las drogas de este tipo alcancen el tumor (1). Un sustrato de Pgp y BCRP es la DOXOrubicina (DOXO), un fármaco utilizado en la terapia anti cáncer, el cual es muy eficaz para atacar las células del tumor cerebral in vitro, pero con un uso clínico limitado por la poca entrega a través de la barrera hematoencefálica (BBB) y por la resistencia propia de los tumores. Por otra parte las células de BBB y las células del tumor cerebral tienen también proteínas superficiales, como el receptor de la lipoproteína de baja densidad (LDLR), que podría utilizarse como blanco terapéutico en BBB y tumores cerebrales. Es asi como la importancia de este estudio se basa en la generación de estrategias terapéuticas que promuevan el paso de las drogas a través de la barrera hematoencefalica y tumoral, y a su vez, se reconozcan mecanismos celulares que induzcan el incremento en la expresión de los transportadores ABC, de manera que puedan ser utilizados como blancos terapéuticos.Este estudio demostró que el uso de una nueva estrategia basada en el “Caballo de Troya”, donde se combina la droga DOXOrubicina, la cual es introducida dentro de un liposoma, salvaguarda la droga de manera que se evita su reconocimiento por parte de los transportadores ABC tanto de la BBB como de las células del tumor. La construcción del liposoma permitió utilizar el receptor LDLR de las células asegurando la entrada a través de la BBB y hacia las células tumorales a través de un proceso de endocitosis. Este mecanismo fue asociado al uso de estatinas o drogas anticolesterol las cuales favorecieron la expresión de LDLR y disminuyeron la actividad de los transportadores ABC por nitración de los mismos, incrementando la eficiencia de nuestro Caballo de Troya. Por consiguiente demostramos que el uso de una nueva estrategia o formulación denominada ApolipoDOXO más el uso de estatinas favorece la administración de fármacos a través de la BBB, venciendo la resistencia del tumor y reduciendo los efectos colaterales dosis dependiente de la DOXOrubicina. Además esta estrategia del "Caballo de Troya", es un nuevo enfoque terapéutico que puede ser considerado como una nueva estrategia para aumentar la eficacia de diferentes fármacos en varios tumores cerebrales y garantiza una alta eficiencia incluso en un medio hipóxico,característico de las células cancerosas, donde la expresión del transportador Pgp se vió aumentada. Teniendo en cuenta la relación entre algunas vías de señalización reconocidas como moduladores de la actividad de Pgp, este estudio presenta no solo la estrategia del Caballo de Troya, sino también otra propuesta terapéutica relacionada con el uso de Temozolomide más DOXOrubicina. Esta estrategia demostró que el temozolomide logra penetrar la BBB por que interviene en la via de señalización de la Wnt/GSK3/β-catenina, la cual modula la expresión del transportador Pgp. Se demostró que el TMZ disminuye la proteína y el mRNA de Wnt3 permitiendo plantear la hipótesis de que la droga al disminuir la transcripción del gen Wnt3 en células de BBB, incrementa la activación de la vía fosforilando la β-catenina y conduciendo a disminuir la β-catenina nuclear y por tanto su unión al promotor del gen mdr1. Con base en los resultados este estudio permitió el reconocimiento de tres mecanismos básicos relacionados con la expresión de los transportadores ABC y asociados a las estrategias empleadas: el primero fue el uso de las estatinas, el cual condujo a la nitración de los transportadores disminuyendo su actividad por la via del factor de transcripción NFκB; el segundo a partir del uso del temozolomide, el cual metila el gen de Wnt3 reduciendo la actividad de la via de señalización de la la β-catenina, disminuyendo la expresión del transportador Pgp. El tercero consistió en la determinación de la relación entre el eje RhoA/RhoA quinasa como un modulador de la via (no canónica) GSK3/β-catenina. Se demostró que la proteína quinasa RhoA promovió la activación de la proteína PTB1, la cual al fosforilar a GSK3 indujo la fosforilación de la β-catenina, lo cual dio lugar a su destrucción por el proteosoma, evitando su unión al promotor del gen mdr1 y por tanto reduciendo su expresión. En conclusión las estrategias propuestas en este trabajo incrementaron la citotoxicidad de las células tumorales al aumentar la permeabilidad no solo de la barrera hematoencefálica, sino también de la propia barrera tumoral. Igualmente, la estrategia del “Caballo de Troya” podría ser útil para la terapia de otras enfermedades asociadas al sistema nervioso central. Por otra parte estos estudios indican que el reconocimiento de mecanismos asociados a la expresión de los transportadores ABC podría constituir una herramienta clave en el desarrollo de nuevas terapias anticáncer.

The antithrombotic effect of dextran-40 in man is due to enhanced fibrinolysis in vivo

BACKGROUND: Dextran-40 is effective in reducing postoperative Doppler-detectable embolization in patients undergoing carotid endarterectomy (CEA). Dextrans are thought to have antithrombotic and antiplatelet effects. The mode of action is unclear. In rats, dextran blocks uptake of tissue plasminogen activator (tPA) by mannose-binding receptors. Because this would have the effect of enhancing endogenous fibrinolysis, we explored this effect of dextran-40 on fibrinolysis in man. METHODS: Twenty patients undergoing endovascular stenting for abdominal aortic aneurysm were randomized to receive 100 mL of 10% dextran-40 or saline, over 1 hour, during their operation in addition to heparin. Blood samples were taken preoperatively, intraoperatively (immediately after operative procedure), and 24 hours postoperatively. Thrombi were formed in a Chandler loop and used to assess endogenous fibrinolysis over 24 hours, measured as the fall in thrombus weight, and the release of fluorescently labelled fibrinogen from the thrombus. Plasma samples were analyzed for markers of fibrinolysis; plasmin-antiplasmin (PAP), PAI-1, and t-PA, and for functional von Willebrand factor (vWF). Platelet response to thrombin and other agonists was measured by flow cytometry. RESULTS: Thrombi formed ex vivo from the intraoperative blood samples from the dextran-treated patients exhibited significantly greater fibrinolysis vs preoperative samples, seen both as a significantly greater percentage reduction in thrombus weight (from 34.7% to 70.6% reduction) and as an 175% increase in the release of fluorescence (P < .05). Fibrinolysis returned to baseline levels the next day. No change was seen in the saline-treated group. Plasma levels of PAP and PAI-1 increased significantly postoperatively in the dextran-treated group vs the saline group (P < .05). The postoperative level of functional VWF was significantly lower in the dextran-treated group vs controls. A specific reduction occurred in the platelet response to thrombin, but not to other agonists, in the intraoperative samples from the dextran-treated group (11.1% vs 37.1%; P = .022), which was not seen in the controls. CONCLUSIONS: These data are consistent with a rise in plasmin due to dextran blockade of tPA uptake in vivo, leading to enhanced fibrinolysis, cleavage of vWF and of the platelet protease-activated receptor-1 (PAR-1) thrombin receptor. This suggests that dextran exerts a combined therapeutic effect, enhancing endogenous fibrinolysis, whilst also reducing platelet adhesion to vWF and platelet activation by thrombin. The proven antithrombotic efficacy of low-dose dextran in carotid surgery may be applicable to wider therapeutic use.

Effect of nωnla, an inhibitor of No-synthase, on the release of tissue-plasminogem activator from rat aorta

Alterations in the synthesis or enhanced inactivation of nitric oxide (NO) and increase in fibrin deposition in the vascular bed lead to an imbalance that can induced intravascular coagulation. NO is produced through L-arginine pathway by constitutive and inducible nitric oxide synthase (NOS). The inducible isoform can be activated by cytokines such as tumor necrosis factor alfa. We evaluated NO-induced tissue-plasminogen activator (t-PA) release from isolated aortic segments of Wistar rats measuring the fibrinolytic activity in the fibrin plate. Inhibition of NO biossynthesis with Nω-nitro-L-arginine (NωNLA) significantly attenuated the fibrinolytic activity (FA) evoked by aortic segments of this group (GII) compared to the saline group (GI). The administration of L-arginine produced restoration of FA in this group (GIII) treated with NωNLA suggesting that t-PA arising from segments of rat aorta is influenced by NO.

uPAR expression in canine normal prostate and with proliferative disorders

Prostatic lesions such as prostatic intraepithelial neoplasia (PIN) and proliferative inflammatory atrophy (PIA) are studied in human and canine species due to their malignance potential. The plasminogen activator (PA) system has been suggested to play a central role in cell adhesion, angiogenesis, inflammation, and tumor invasion. The urokinase-type plasminogen activator receptor (uPAR) is a component of the PA, with a range of expression in tumor and stromal cells. In this study, uPAR expression in both canine normal prostates and with proliferative disorders (benign prostatic hyperplasia-BPH, proliferative inflammatory atrophy-PIA, prostatic intraepithelial neoplasia-PIN, and carcinoma-PC) was evaluated by immunohistochemistry in a tissue microarray (TMA) slide to establish the role of this enzyme in extracellular matrix (ECM) remodeling and in the processes of tissue invasion. A total of 298 cores and 355 diagnoses were obtained, with 36 (10.1%) normal prostates, 46 (13.0%) with BPH, 128 (36.1%) with PIA, 74 (20.8%) with PIN and 71 (20.0%) with PC. There is variation in the expression of uPAR in canine prostate according to the lesion, with lower expression in normal tissue and with BPH, and higher expression in tissue with PIA, PIN and PC. The high expression of uPAR in inflammatory and neoplastic microenvironment indicates increased proteolytic activity in canine prostates with PIA, PIN, and PC.

Die funktionelle Rolle von LRP1 in der tPA-vermittelten Aktivierung von NMDA-Rezeptoren

Das Low Density Lipoprotein Receptor-related Protein 1 (LRP1) scheint neben seiner ursprünglichen Rolle als Lipoproteinrezeptor auch eine fundamentale Rolle bei der Einleitung von Signaltransduktionskaskaden im sich entwickelnden Gehirn zu spielen. Einer seiner Hauptliganden ist die Serinprotease Tissue-type Plasminogen Aktivator (tPA), welche NMDA-Rezeptor-abhängig MAP Kinasenaktivierung induzieren kann. In dieser Studie sollte daher untersucht werden, ob LRP1 und der NMDA Rezeptor in der tPA-vermittelten Signaltransduktion miteinander kooperieren. Es konnte gezeigt werden, dass sowohl LRP1 als auch der NMDA Rezeptor an der tPA-induzierten Erk1/2 Phosphorylierung beteiligt sind, da dieser Effekt mit den spezifischen Inhibitoren RAP, MK-801 und DL-AP5 blockiert werden konnte. Eine weitere Bestätigung der LRP1-Spezifität zeigte sich durch shRNA knock-down Experimente. Calcium Imaging Experimente ergaben, dass die Applikation von tPA sowohl in primären, hippokampalen Neuronen als auch in der neuronalen Zelllinie HT22 zu einem robusten Einstrom von Calcium in die Zelle führte, welcher mit dem NMDA Rezeptor Inhibitor MK-801 und dem LRP1 Inhibitor RAP blockiert werden konnte. RNAi Experimente und Überexpressionsstudien bestätigten die Beteiligung von PSD-95 als intrazelluläres Adapterprotein, welches die beiden Rezeptoren miteinander verbindet. Als Bindungsstelle für PSD-95 konnte mit Hilfe von LRP1 knock-in Mausneuronen die distale NPxY(2) Domäne am LRP1 C-Terminus identifiziert werden. Diese Ergebnisse führten zu der Hypothese eines multimeren tPA-LRP1-NMDA Rezeptor Komplexes, der über die primäre Bindung von tPA an LRP1 aktiviert wird und anschließend das Signal an den NMDA Rezeptor weiterleitet. Somit weisen die Ergebnisse dieser Arbeit auf einen neuen, tPA-vermittelten Mechanismus zur Öffnung von Glutamatrezeptoren hin, der eine funktionelle Kooperation von dem Lipoproteinrezeptor LRP1 mit dem NMDA Rezeptor voraussetzt.