Human tissue-type plasminogen activator.

Tissue-type plasminogen activator (t-PA ) plays an important role in the removal of intravascular fibrin deposits and has several physiological roles and pathological activities in the brain. Its production by many other cell types suggests that t-PA has additional functions outside the vascular and central nervous system. Activity of t-PA is regulated at the level of its gene transcription, its mRNA stability and translation, its storage and regulated release, its interaction with cofactors that enhance its activity, its inhibition by inhibitors such as plasminogen activator inhibitor type 1 or neuroserpin, and its removal by clearance receptors. Gene transcription of t-PA is modulated by a large number of hormones, growth factors, cytokines or drugs and t-PA gene responses may be tissue-specific. The aim of this review is to summarise current knowledge on t-PA function and regulation of its pericellular activity, with an emphasis on regulation of its gene expression.

Ex vivo pulsatile perfusion of human saphenous veins induces intimal hyperplasia and increased levels of the plasminogen activator inhibitor 1.

Vessel wall trauma induces vascular remodeling processes including the development of intimal hyperplasia (IH). To assess the development of IH in human veins, we have used an ex vivo vein support system (EVVSS) allowing the perfusion of freshly isolated segments of saphenous veins in the presence of a pulsatile flow which reproduced arterial conditions regarding shear stress, flow rate and pressure during a period of 7 and 14 days. Compared to the corresponding freshly harvested human veins, histomorphometric analysis showed a significant increase in the intimal thickness which was already maximal after 7 days of perfusion. Expression of the endothelial marker CD31 demonstrated the presence of endothelium up to 14 days of perfusion. In our EVVSS model, the activity as well as the mRNA and protein expression levels of plasminogen activator inhibitor 1, the inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), were increased after 7 days of perfusion, whereas the expression levels of tPA and uPA were not altered. No major change was observed between 7 and 14 days of perfusion. These data show that our newly developed EVVSS is a valuable setting to study ex vivo remodeling of human veins submitted to a pulsatile flow.

Characterization of endothelial-like cells derived from human mesenchymal stem cells.

BACKGROUND: Blood-derived endothelial progenitor cells (EPC) have been used to treat ischemic disease. However, the number of EPC that can be obtained from adult blood is limited. OBJECTIVE: To characterize endothelial-like cells obtained from human bone marrow and determine their ability to stimulate new blood vessel formation in vivo. METHODS: Mononuclear cells (MNC) were isolated from human bone marrow or umbilical cord blood and cultured in endothelial growth medium (EGM-2). Mesenchymal stem cells (MSC) were isolated from bone marrow and induced to differentiate into endothelial-like cells (MSCE), or adipocytes or osteocytes by growth in EGM-2, adipogenic or osteogenic medium. RESULTS: Cells obtained by culturing bone marrow MNC in EGM-2 formed cord- or tube-like structures when grown on Matrigel(TM) and expressed several endothelial marker proteins. However, cell morphology and the profile of endothelial marker protein expression were different from those of cord blood-derived EPC (cbEPC). Cells with a similar phenotype were obtained by differentiation of MSC into MSCE, which was accompanied by an increase of endothelial marker proteins and a diminished capacity to differentiate into adipocytes. Subcutaneous implantation of MSCE in collagen plugs in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice resulted in formation of functional blood vessels that had incorporated the MSCE. CONCLUSIONS: Our results show that MSCE and cbEPC are different cell types. The formation of functional blood vessels by MSCE, combined with high yields and a reduced capacity to differentiate into other cell types compared with MSC, makes these cells potentially useful for autologous therapy of ischemic disease.

Aspirin inhibits endothelial cell activation induced by antiphospholipid antibodies.

BACKGROUND: Antiphospholipid antibodies (APLA) have been shown to activate endothelial cells (EC) in vitro, as documented by an increased expression of tissue factor as well as leukocyte adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin. Currently, treatment of patients with the antiphospholipid syndrome includes aspirin, particularly for women with recurrent fetal loss. OBJECTIVE: The present study was undertaken to investigate whether aspirin interferes with EC activation induced by APLA in vitro. METHODS: IgG from 14 patients with APLA, and suffering from thrombotic complications and/or pregnancy morbidity, and control IgG were tested for their ability to modify the expression of VCAM-1 in human umbilical vein endothelial cells. VCAM-1 antigen was measured by flow cytometry and its mRNA by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Incubation of EC with IgG from most of the patients led to a higher VCAM-1 expression compared with incubation with control IgG. The effect of aspirin was studied for the eight IgG samples that induced a more than 50% increase in VCAM-1. Aspirin (10 mm) treatment of the cells significantly reduced the VCAM-1 response to these APLA. CONCLUSIONS: Our results indicate that besides its antiplatelet properties, aspirin exerts a protective effect towards APLA at the EC level by decreasing leukocyte adhesion molecule expression at the cell surface.

Effects of statins on adhesion molecule expression in endothelial cells.

BACKGROUND: Inhibitors of HMG-CoA reductase are widely used to prevent atherosclerosis progression. The expression of adhesion molecules on activated endothelial cells (EC) is an important step in the initiation and progression of atherosclerosis. OBJECTIVES: We investigated whether adhesion molecule expression on activated EC is influenced by simvastatin, fluvastatin and pravastatin and, if so, by which mechanisms. METHODS: Human EC from umbilical veins or saphenous veins were pretreated overnight with statins with or without mevalonate, and also for simvastatin or fluvastatin with the isoprenoid intermediates, farnesyl pyrophosphate (FPP), or geranylgeranyl pyrophosphate (GGPP). After 4-6 h activation with tumor necrosis factor (TNF)-alpha or lipopolysaccharide (LPS), surface adhesion molecule expression was evaluated by ELISA and by flow cytometry. The same experiments were performed with selective inhibitors of geranylgeranyltransferase (GGTI-286) and farnesyltransferase (FTI-277). RESULTS: Pretreatment with simvastatin, fluvastatin or pravastatin potentiated the TNF-alpha and LPS-induced expression of E-selectin and VCAM-1, and mevalonate reversed the potentiating effect of these statins. GGPP also reversed the potentiating effect of simvastatin or fluvastatin on adhesion molecule expression, while FPP only partially reversed this effect. Furthermore, GGTI-286, but not FTI-277, mimicked the effect of simvastatin by increasing the TNF-alpha-mediated overexpression of E-selectin. CONCLUSIONS: Statins increase E-selectin- and VCAM-1-induced expression on vascular endothelial cells stimulated with TNF-alpha or LPS. The inhibition of geranylgeranylated proteins could contribute to this effect.

Localization of beta2-glycoprotein 1 in late endosomes of human endothelial cells.

Antiphospholipid antibodies (APLA) are associated with thrombophilia and recurrent pregnancy loss. Different mechanisms have been proposed to explain their pathogenic effects and among them, we have previously shown that APLA accumulate in late endosomes of human umbilical vein endothelial cells (HUVEC) leading to a redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR). Because many APLA are directed towards beta2-glycoprotein 1 (beta2GP1)phospholipid complexes, we investigated the localisation of beta2GP1 in HUVEC. By immunofluorescence analysis, using monoclonal and polyclonal anti-beta2GP1 antibodies, we detected beta2GP1 at the cell surface and in late endosomes. Incubation of HUVEC with anti-beta2GP1 antibodies resulted in antibody accumulation at the cell surface and within late endosomes and in a redistribution of the CI-M6PR from the Golgi apparatus to late endosomes. The anti-beta2GP1 antibodies remained detectable in late endosomes even after several days of incubation in antibody-free medium. The accumulation of anti-beta2GP1 antibodies in late endosomes of endothelial cells and the resulting modification of intracellular protein trafficking may contribute to the pathogenic effects of these antibodies.

Mycological control and surveillance of biological waste and compost.

This paper presents some recent developments regarding current work on hygienic aspects, in particular the presence and dispersion of fungi (e.g. Aspergillus fumigatus), of biological waste and compost.

PAF acether release on antigenic challenge. A method for the investigation of drug allergic reactions.

This study describes an in vitro method to evaluate a PAF acether release test (PART) from white blood cells after antigenic challenge. PAF acether activity of the supernatant was tested by platelet aggregation. The aggregating power was abolished by using SRI 63-441 (Sandoz), a PAF acether inhibitor. This method was applied to 57 patients with allergic or pseudo-allergic reactions to drugs by using different drug protein conjugates. The results of PART were evaluated in relation to the clinical history (score of imputability) and to other tests (skin tests, lymphocyte transformation tests (LTT), IgE-RAST). A good correlation was found between the release of PAF acether and a high predictability score: sensitivity 75%, specificity 83.8%. PART also correlated with skin tests (75% agreement, n = 60), with LTT (67.7% agreement, n = 74) and in 65.6% of cases with positive penicillin IgE-RAST (n = 32). This method brings a new possibility for the investigation of drug-allergic and pseudo-allergic reactions.

Comparative analysis of lyophilized fresh fruits and vegetables with commercial extracts for skin testing of patients with food allergy.

Most allergic reactions to fruits and vegetables are immediate IgE-mediated reactions following either contact or ingestion. In such problems, the practice of skin tests represents an important diagnostic tool. However, we often observe a discordance between the clinic and the results of the skin tests performed with commercial glycerinated extracts. A simple procedure of lyophilization applied to some fruits and vegetables is described, which allowed the comparison with fresh juicy material and commercial extracts. This material was applied in 10 individuals shown to be allergic to the corresponding food and in 10 healthy controls. When compared to the commercial extracts, the lyophilized material showed a significantly better sensitivity and specificity. Some control individuals reacted, but insignificantly, to the lyophilized material. The technical procedure is very simple. It allows the preparation of large amounts of aliquot, kept at -20 degrees C until needed. Further work is needed to extend the procedure to other allergens and to improve the standardization.