Jordi Félez

UV Irradiation Induces the Murine Urokinase-Type Plasminogen Activator Gene via the c-Jun N-Terminal Kinase Signaling Pathway: Requirement of an AP1 Enhancer Element

ABSTRACT UV irradiation leads to severe damage, such as cutaneous inflammation, immunosuppression, and cancer, but it also results in a gene induction protective response termed the UV response. The signal triggering the UV response was thought to originate from DNA damage; recent findings, however, have shown that it is initiated at or near the cell membrane and transmitted via cytoplasmic kinase cascades to induce gene transcription. Urokinase-type plasminogen activator (uPA) was the first protein shown to be UV inducible in xeroderma pigmentosum DNA repair-deficient human cells. However, the underlying molecular mechanisms responsible for the induction were not elucidated. We have found that the endogenous murine uPA gene product is transcriptionally upregulated by UV in NIH 3T3 fibroblast and F9 teratocarcinoma cells. This induction required an activator protein 1 (AP1) enhancer element located at −2.4 kb, since deletion of this site abrogated the induction. We analyzed the contribution of the three different types of UV-inducible mitogen-activated protein (MAP) kinases (ERK, JNK/SAPK, and p38) to the activation of the murine uPA promoter by UV. MEKK1, a specific JNK activator, induced transcription from the uPA promoter in the absence of UV treatment, whereas coexpression of catalytically inactive MEKK1(K432M) and of cytoplasmic JNK inhibitor JIP-1 inhibited UV-induced uPA transcriptional activity. In contrast, neither dominant negative MKK6 (or SB203580) nor PD98059, which specifically inhibit p38 and ERK MAP kinase pathways, respectively, could abrogate the UV-induced effect. Moreover, our results indicated that wild-type N-terminal c-Jun, but not mutated c-Jun (Ala-63/73), was able to mediate UV-induced uPA transcriptional activity. Taken together, we show for the first time that kinases of the JNK family can activate the uPA promoter. This activation links external UV stimulation and AP1-dependent uPA transcription, providing a transcription-coupled signal transduction pathway for the induction of the murine uPA gene by UV.

Characterization of tissue factor expression on the human endothelial cell line ECV304

The endothelial cell line ECV304 is a spontaneously transformed cell line established from human umbilical vein. The characterization of tissue factor (TF) expression by ECV304 cells has been accomplished in this study. ECV304 cells expressed both TF mRNA and antigen (TFag) constitutively. In ECV304 cell lysates, the levels of TFag (1.4 ± 0.3 ng of TFag/106 cells) were considerably higher than in THP‐1 monocytoid cells (0.07 ± 0.03 ng of TFag/106 cells). TFag was also detected on the ECV304 cell surface by flow cytometric studies. In binding analyses, 3.5 ± 0.7 × 104 molecules of TF per cell were estimated, similar to the amounts found in ECV304 cell lysates (2.9 ± 0.6 × 104 molecules/cell), suggesting that all TFag was translocated to the cell surface. Phorbol myristate acetate (PMA) stimulation of ECV304 cells resulted in an increase of TF mRNA levels, which was abrogated when gene transcription was impaired, suggesting a transcriptional regulation of the TF gene by PMA. In contrast, TFag was not elevated by PMA‐stimulation, indicating the existence of additional posttranscriptional mechanisms. Thus, ECV304 cells constitute a singular endothelial cell model for exploring the regulation of TF expression. Am. J. Hematol. 56:71–78, 1997.

Mutations in HAMP and HJV genes and their impact on expression of clinical hemochromatosis in a cohort of 100 Spanish patients homozygous for the C282Y mutation of HFE gene

Most hereditary hemochromatosis (HH) patients are homozygous for the C282Y mutation of the HFE gene. Nevertheless, penetrance of the disease is very variable. In some patients, penetrance can be mediated by concomitant mutations in other iron master genes. We evaluated the clinical impact of hepcidin (HAMP) and hemojuvelin mutations in a cohort of 100 Spanish patients homozygous for the C282Y mutation of the HFE gene. HAMP and hemojuvelin mutations were evaluated in all patients by bidirectional direct cycle sequencing. Phenotype–genotype interactions were evaluated. A heterozygous mutation of the HAMP gene (G71D) was found in only one out of 100 cases. Following, we performed a study of several members of that family, and we observed several members had a digenic inheritance of the C282Y mutation of the HFE gene and the G71D mutation of the HAMP gene. This mutation in the HAMP gene did not modify the phenotype of the individuals who were homozygous for the C282Y mutation. One other patient presented a new polymorphism in the hemojuvelin gene, without consequences in iron load or clinical course of the disease. In conclusion, HAMP and hemojuvelin mutations are rare among Spanish HH patients, and their impact in this population is not significant.

Monoclonal antibodies detect receptor-induced binding sites in Glu-plasminogen

When Glu-plasminogen binds to cells, its activation to plasmin is markedly enhanced compared with the reaction in solution, suggesting that Glu-plasminogen on cell surfaces adopts a conformation distinct from that in solution. However, direct evidence for such conformational changes has not been obtained. Therefore, we developed anti-plasminogen mAbs to test the hypothesis that Glu-plasminogen undergoes conformational changes on its interaction with cells. Six anti-plasminogen mAbs (recognizing 3 distinct epitopes) that preferentially recognized receptor-induced binding sites (RIBS) in Glu-plasminogen were obtained. The mAbs also preferentially recognized Glu-plasminogen bound to the C-terminal peptide of the plasminogen receptor, Plg-R(KT), and to fibrin, plasmin-treated fibrinogen, and Matrigel. We used trypsin proteolysis, immunoaffinity chromatography, and tandem mass spectrometry and identified Glu-plasminogen sequences containing epitopes recognized by the anti-plasminogen-RIBS mAbs: a linear epitope within a domain linking kringles 1 and 2; a nonlinear epitope contained within the kringle 5 domain and the latent protease domain; and a nonlinear epitope contained within the N-terminal peptide of Glu-plasminogen and the latent protease domain. Our results identify neoepitopes latent in soluble Glu-plasminogen that become available when Glu-plasminogen binds to cells and demonstrate that binding of Glu-plasminogen to cells induces a conformational change in Glu-plasminogen distinct from that of Lys-Pg.

Detection of hereditary hemochromatosis and biochemical iron overload in primary care: A multicenter case finding study in Spain†

Although the C282Y HFE gene substitution is the most common mutation among individuals from Northern Europe in Spain and Italy, this mutation has a lower prevalence and thus a lower proportion of Spanish and Italian patients are C282Y homozygotes in comparison to Northern European descendants. These differences prompted us to explore the expression of hereditary hemochromatosis (HH) in a set of Spanish individuals in primary care settings. We undertook a multicenter prospective case-finding study including 2,739 individuals. Subjects with consistent elevated transferring saturation and/or serum ferritin levels were referred has having elevated iron measures (EIM), and molecular analyses for HFE-gene were performed. EIM were consistently found in 126 subjects. Among them 14 HH patients were identified (3 C282Y homozygotes and 11 compound C282Y/H63D heterozygotes), including subjects from familial studies. A hitherto new mutation of HFE was detected (V284M/H63D) as well as a family carrying a hereditary hyperferritinemia/cataract syndrome (HHCS) with a C39T mutation in L-ferritin gene. A 5-year follow-up of patients with HH showed a steady state of the iron measurements as well as in their clinical situation. Forty-five percent of patients with EIM had metabolic syndrome. This is the first case-finding study performed in Spain on these topics.

Characterization of Plasminogen Binding to NB4 Promyelocytic Cells Using Monoclonal Antibodies against Receptor-Induced Binding Sites in Cell-Bound Plasminogen

The NB4 promyelocytic cell line exhibits many of the characteristics of acute promyelocytic leukemia blast cells, including the translocation (15 : 17) that fuses the PML gene on chromosome 15 to the RARα gene on chromosome 17. These cells have a very high fibrinolytic capacity. In addition to a high secretion of urokinase, NB4 cells exhibit a 10-fold higher plasminogen binding capacity compared with other leukemic cell lines. When tissue-type plasminogen activator was added to acid-treated cells, plasmin generation was 20–26-fold higher than that generated by U937 cells or peripheral blood neutrophils, respectively. We found that plasminogen bound to these cells can be detected by fluorescence-activated cell sorting using an antiplasminogen monoclonal antibody that specifically reacts with this antigen when it is bound to cell surfaces. All-trans retinoid acid treatment of NB4 cells markedly decreased the binding of this monoclonal antibody. This cell line constitutes a unique model to explore plasminogen binding and activation on cell surfaces that can be modulated by all-trans retinoid acid treatment.

Nurse-Driven Training Courses: Impact on Implementation of Ambulatory Blood Pressure Monitoring

Background: Ambulatory blood pressure monitoring (ABPM) predicts cardiovascular risk and identifies white-coat and masked hypertension, efficacy of treatment and the circadian cycle of hypertensive patients. Objective: To analyze the effectiveness of ABPM implementation thoughtout a nurse-driven training program. Materials and Methodology: Twenty eight professionals were involved in the study carried out in the primary care center of the metropolitan area of Barcelona that serves 34,289 inhabitants. The ABPM implementation program was driven by two nurses that held four education sessions. After a 2-year follow-up period, we assessed the outcome of attendance at the educational sessions. First, we evaluated whether the program increased the number of orders of ABPM. Second, we used a survey to evaluate to what extent the input of our educational sessions was understood by attendants. Third, we analyzed the effect ABPM results had on the treatment of patients with a bad control of their hypertension. Results: After the training sessions we found a 6-fold increase in the number of patients undergoing ABPM. We analyzed 204 hypertensive individuals: 41% dippers, 34% were non-dippers, 20% were risers and 5% were extremely dippers. According to our survey, 100% of attendants had a good practice regarding ABPM management. However only 27% of riser patients were studied with a second ABPM. Conclusions: Specific training processes are needed for implementation of ABPM and an even more concentrated effort should be focused on training in the correct interpretation of ABPM results.

Monoclonal antibodies against receptor-induced binding sites detect cell-bound plasminogen in blood

Binding of Glu-plasminogen (the native, circulating form of the zymogen) to cells results in enhancement of its activation. Cell-associated plasmin proteolytic activity is a key component of physiologic and pathologic processes requiring extracellular matrix degradation. Recently, we developed antiplasminogen mAbs that recognize receptor-induced binding sites (RIBS) in Glu-plasminogen and, therefore, preferentially react with cell-associated Glu-plasminogen in the presence of soluble Glu-plasminogen. Here we have used FACS with a representative antiplasminogen receptor-induced binding site mAb, mAb49, to examine whether plasminogen associates with peripheral blood cells in blood. Plasminogen binding to neutrophils, monocytes, B-lymphocytes, T-lymphocytes, and platelets was clearly detected. Treatment of whole blood with lipopolysaccharide or 12-0 tetradecanoylphorbol-13-acetate up-regulated plasminogen binding to neutrophils and in vivo treatment with all-trans retinoic acid decreased plasminogen binding to acute promyelocytic leukemia blasts. Our results demonstrate that mAb49 can be used to monitor cell-bound plasminogen in blood under both normal and pathologic conditions.

Assessment of primary healthcare professionals’ management of hypertensive patients with riser pattern

Background: Ambulatory blood pressure monitoring (ABPM) was implemented in our primary care setting four years ago. Since then, 450 ABPMs have been performed and 69 riser subjects identified. The riser pattern is an independent risk factor for both incidence of cardiovascular events and their associated mortality. Objective: The purpose of this study was to assess the amount of control of essential hypertension (EH) among riser patients and to evaluate how our health professionals manage therapeutic changes in riser individuals. Materials and methodology: This retrospective study involved 34,289 inhabitants served in a centre in the Barcelona metropolitan area. EH individuals (450) were recruited and ABPM was performed following guidelines of the MAPAPRES (www.cardiorisc.com/MP/index_MP.asp). Results: Good control of blood pressure was observed in 46% of dipper and non-dipper subjects but only 35% of riser subjects had blood pressures within good control ranges. The measured cardiovascular risk was either high or very high in 35% of riser individuals. Changes in medication were introduced in riser patients with both good and poor blood pressure control. A second follow-up ABPM was done in only 27% of the riser individuals. In these subjects, therapeutic changes successfully modified ABPM patterns in 87% of cases. Conclusions: Therapeutic changes in riser patients were introduced when these subjects were poorly controlled and these changes were highly effective. Additional ABPM to confirm the effectiveness of therapeutic changes was only performed in some individuals. Thus, for management of riser patients, more specific training of health professionals is needed.

The Role of Lys-Plasminogen in Cell-Mediated Plasmin Production

A key control point by which the activity of plasmin is positively regulated is by localizing Glu-plasminogen (the native circulating form of the plasminogen molecule) to specific binding sites on cell surfaces. Glu-plasminogen is activated much more efficiently when bound to cells than when in solution. Upon activation, plasmin remains associated with the cell surface where plasmin activity is relatively protected from inhibitors. Furthermore, the enzymatic activity of plasmin is enhanced in the milieu of the cell surface.