Effects of preadipocytes derived from mice fed with high fat diet on the angiogenic potential of endothelial cells.

BACKGROUND AND AIMS: Obesity promotes a persistent inflammatory process in the adipose tissue, activating the endothelium and leading to vascular dysfunction. Preadipocytes can interact with endothelial cells in a paracrine way stimulating angiogenesis. However, the potential of preadipocytes from adipose tissue of high fat diet (HFD) fed animal to stimulate angiogenesis has not been evaluated yet. The aim of this study was to investigate the effects of such diet on the angiogenic potential of preadipocytes in a mice model. METHODS AND RESULTS: We have evaluated body weight gain, fasting glucose levels and insulin resistance, mRNA expression in preadipocytes and endothelial cells after co-culture with preadipocytes, in vivo vascular function and in vitro endothelial cell migration and tubulogenesis. High fat diet promoted an increase in body weight, glycemic index and insulin resistance in mice. Preadipocytes mRNA expression of factors involved in angiogenesis was higher in these animals. In endothelial tEnd cells mRNA expression of factors involved in vessel growth were higher after co-culture with preadipocytes derived from mice fed with HFD. Although no significant differences were observed in in vivo vasodilatation response between control and HFD groups, endothelial tEnd cells showed an increase in migration and tubulogenesis when cultivated with conditioned media from preadipocytes derived from mice fed with HFD. CONCLUSION: Hypoxic and growth factors produced by preadipocytes derived from mice fed with HFD have higher capacity than preadipocytes derived from mice fed with standard diet to stimulate the angiogenic potential of endothelial cells, contributing to vascular disorders in obesity.

Development of asymmetric resorbable membranes for guided bone and surrounding tissue regeneration.

Membranes design for guided tissue engineering have been studied to aid in cell viability and function as tissue barriers. Two asymmetric resorbable membranes for guided bone regeneration (GBR) were produced: chitosan/pectin/poly-caprolactone (PECm) and poly(vinyl alcohol)/polyethylenimine/poly(ethylene glycol) (PVAm). Both membranes were characterized by physical, chemical, mechanical, degradation rate, and in vitro biological assessment. Scanning electron microscopy (SEM) confirmed the membranes asymmetry, in which PECm asymmetry is given by roughness and chemical composition, while PVAm's only by differences in porosity. Fourier transform infrared spectroscopy (FTIR) identified chemical groups and bonds between polymers. Both sides of PVAm revealed to be hydrophobic, whereas the PECm presented one side with higher hydrophobicity than the other. In vitro biological assessment disclosed that PECm presented a higher cell adhesion growth pattern than PVAm, where it seemed to occur a delay in proliferation due to initial low cell adhesion. Both developed membranes are suitable for GBR, since both membranes fulfil the requirements to be used as a tissue barrier. The PECm has an additional role in cell viability that was not observed in the PVAm. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2141-2150, 2018.

Lipoxin A4 selectively programs the profile of M2 tumor-associated macrophages which favour control of tumor progression.

In tumor microenvironments, the macrophage population is heterogeneous, but some macrophages can acquire tumor-promoting characteristics. These tumor-associated macrophages (TAM) exhibit an M2-like profile, with deficient production of NO and ROS, characteristics of pro-inflammatory M1 cytotoxic macrophages. Lipoxins (LX) and 15-epi-lipoxins are lipid mediators which can induce certain features of M2 macrophages in mononuclear cells, but their effects on TAM remain to be elucidated. This study tested the hypothesis that ATL-1, a synthetic analogue of 15-epi-lipoxin A4 , could modulate TAM activity profile. We show that human macrophages (MΦ) differentiated into TAM-like cells after incubation with conditioned medium from MV3, a human melanoma lineage cell. Contrasting with the effects observed in other M2 subsets and M1 profile macrophages, ATL-1 selectively decreased M2 surface markers in TAM, suggesting unique behavior of this particular M2 subset. Importantly, these results were replicated by the natural lipoxins LXA4 and the aspirin induced 15-epi-LXA4 (ATL). In parallel, ATL-1 stimulated TAM to produce NO by increasing the iNOS/arginase ratio and activated NADPH oxidase, triggering ROS production. These alterations in TAM profile induced by ATL-1 led to loss of the anti-apoptotic effects of TAM on melanoma cells and increased their cytotoxic properties. Finally, ATL-1 was found to inhibit tumor progression in a murine model in vivo, which was accompanied by alterations in TAM profile and diminished angiogenesis. Together, the results show an unexpected effect of lipoxin, which induces in TAM a change from an M2- to an M1-like profile, thereby triggering tumor cell apoptosis and down-modulating the tumor progression.

Recurrent acute thermal lesion induces esophageal hyperproliferative premalignant lesions in mice esophagus.

Hot beverage consumption is a risk factor for esophageal squamous cell carcinoma, but the underlying mechanisms are still unknown. We developed an experimental mouse model to understand the mechanism of thermal lesion to esophageal carcinogenesis. Female BALB/c mice were treated by gavage with water at different temperatures three times a week and nitrosamines in the drinking water. Water at 70°C, but not at lower temperatures, initially induced an esophageal necrosis that healed and became resistant to necrosis after further administrations. However, when 70°C water was associated with N-nitrosodiethylamine at doses above 1 ppm, there was interference in epithelial regeneration, allowing recurrent thermal injury and inflammation. Recurrent thermal injury resulted in hyper proliferative premalignant lesions being induced earlier (at 4 weeks) and at a higher frequency (4-fold increase at 16 weeks) when compared to mice treated with NDEA only. Ki-67 immunostaining revealed that recurrent thermal injury induced basal cell proliferation resulting in the expansion of epithelial basal cells, confirmed by the increase in cytokeratin 14 positive cells with concomitant reduction of differentiated cytokeratin 5 positive cells. We conclude that recurrent thermal lesion may act as a tumor promoter though a strong proliferation stimulus of esophageal epithelial basal cells.

Effect of selective and non-selective serotonin receptor activation on L-DOPA-induced therapeutic efficacy and dyskinesia in parkinsonian rats.

Selective activation of 5-HT1 receptors has been shown to produce near to full suppression of L-DOPA-induced dyskinesia (LID) in animal models of Parkinson's disease; however, a reduction of the therapeutic effect of L-DOPA has been reported in several studies. Conversely, we recently found that increasing the serotonergic tone with chronic administration of the serotonin precursor 5-hydroxy-tryptophan (5-HTP) can reduce LID in 6-OHDA-lesioned rats, without affecting L-DOPA efficacy. To directly compare the effects of selective versus non-selective serotonin receptor activation, here we first tested different acute doses of the 5-HT1A/1B receptor agonist eltoprazine and 5-HTP on LID in order to identify doses of the individual compounds showing similar anti-dyskinetic efficacy in L-DOPA-primed dyskinetic rats. About 50% reduction of LID was observed with 0.1 mg/kg and 24 mg/kg of eltoprazine and 5-HTP, respectively; we then compared the effect of the two drugs, individually and in combination, on L-DOPA-induced stepping test in L-DOPA-naïve parkinsonian animals and LID over three weeks of L-DOPA treatment. Results showed that eltoprazine induced significant worsening of L-DOPA-mediated performance in the stepping test, while 5-HTP did not. Interestingly, combination of 5-HTP with eltoprazine prevented the reduction in the forelimb use induced by eltoprazine. Moreover, 5-HTP and eltoprazine given individually showed similar efficacy also upon chronic treatment, and had additive effect in dampening the appearance of LID when given in combination. Finally, chronic administration of eltoprazine and/or 5-HTP did not affect striatal serotonin innervation, compared to l-DOPA alone, as measured by serotonin transporter expression.

How demanding is the brain on a reversal task under day and night conditions?

Reversal learning has been studied as the process of learning to inhibit previously rewarded actions. These behavioral studies are usually performed during the day, when animals are in their daily period rest. However, how day or night affects spatial reversal learning and the brain regions involved in the learning process are still unknown. We conducted two experiments using the Morris Water Maze under different light-conditions: naïve group (CN, n=8), day group (DY, n=8), control DY group (CDY, n=8) night group (NG, n=8), and control NG group (CNG, n=7). Distance covered, velocity and latencies to reach the platform were examined. After completing these tasks, cytochrome c-oxidase activity (CO) in several brain limbic system structures was compared between groups. There were no behavioral differences in the time of day when the animals were trained. However, the metabolic brain consumption was higher in rats trained in the day condition. This CO increase was supported by the prefrontal cortex, thalamus, dorsal and ventral striatum, hippocampus and entorhinal cortex, revealing their role in the performance of the spatial reversal learning task. Finally, the orbitofrontal cortex has been revealed as a key structure in reversal learning execution.

Effect of serotonin transporter blockade on L-DOPA-induced dyskinesia in animal models of Parkinson's disease.

Serotonin transporter blockade with selective serotonin reuptake inhibitors (SSRIs) was recently shown to counteract L-DOPA-induced dyskinesia in 6-hydroxydopamine (6-OHDA)-lesioned rats. However, this effect has never been described in Parkinson's disease (PD) patients, despite that they often receive SSRIs for the treatment of depression. In the present study, we investigated the efficacy of the SSRI citalopram against dyskinesia in two experimental models of PD, the 6-OHDA-lesioned rat and 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine (MPTP)-treated macaque. First, we studied the acute and chronic effect of citalopram, given at different time points before L-DOPA, in L-DOPA-primed parkinsonian rats. Moreover, the acute effect of citalopram was also evaluated in dyskinetic MPTP-treated macaques. In L-DOPA-primed rats, a significant and long-lasting reduction of L-DOPA-induced dyskinesia (LID) was observed only when citalopram was given 30 min before L-DOPA, suggesting that the time of injection relative to L-DOPA is a key factor for the efficacy of the treatment. Interestingly, an acute challenge with the 5-HT1A/1B receptor agonist eltoprazine, given at the end of the chronic study, was equally effective in reducing LID in rats previously chronically treated with L-DOPA or L-DOPA plus citalopram, suggesting that no auto-receptor desensitization was induced by chronic citalopram treatment. In MPTP-treated macaques, citalopram produced a striking suppression of LID but at the expense of L-DOPA therapeutic efficacy, which represents a concern for possible clinical application.

Calcificaciones esclerocoroideas asociadas a hipovitaminosis D / Sclerochoroidal calcification associated with hypovitaminosis D

CASO CLÍNICO: Mujer de 69 años que presenta en una exploración funduscópica rutinaria unas lesiones subretinianas blanco-amarillentas en la periferia media temporal superior de ambos ojos. Ecográficamente eran hiperecogénicas, situándose a nivel esclerocoroideo. Se realizó una tomografía axial computarizada que mostró calcificaciones esclerocoroideas posterolaterales. El estudio metabólico reveló una deficiencia severa de vitamina D sin otros hallazgos significativos. Discusión: Las calcificaciones esclerocoroideas son lesiones poco frecuentes que se producen como consecuencia del depósito de calcio a nivel de la esclera y la coroides. Tienen un aspecto clínico característico y en la mayor parte de los casos son idiopáticas. En ocasiones se asocian a enfermedades que cursan con alteraciones del metabolismo del calcio y fósforo, por lo que es necesario realizar un estudio metabólico completo. Se presenta un caso de calcificaciones esclerocoroideas bilaterales asociadas a una hipovitaminosis D de grado severo sin otros hallazgos metabólicos

CASE REPORT: A 69 year-old woman was referred for a routine visit, during which funduscopy revealed white-yellow subretinal lesions in the superotemporal mid-periphery of both eyes. A and B scan ultrasound showed hyperechogenic lesions located at scleral and choroidal level. Computed tomography revealed posterolateral sclerochoroidal calcifications. Metabolic studies showed a severe vitamin D deficiency with no other remarkable findings. Discussion: Sclerochoroidal calcifications are an infrequent finding that occur as a result of calcium deposit at scleral and choroidal level. They have a characteristic clinical picture and are idiopathic in most cases, but may be associated with some systemic diseases, such as calcium and phosphorous metabolic disorders; this fact warrants a thorough metabolic study. We report a case of bilateral sclerochoroidal calcifications associated with severe vitamin D deficiency with no other significant metabolic findings

ATL-1, a synthetic analog of lipoxin, modulates endothelial permeability and interaction with tumor cells through a VEGF-dependent mechanism.

Lipoxins (LX) and 15-epi-LX are lipids with a potent inhibitory effect on angiogenesis, in different models in vivo and in vitro. ATL-1, a synthetic analog of 15-epi-LXA4, inhibits various actions stimulated by vascular endothelial growth factor (VEGF). However, LX actions on endothelial cells (EC) in tumor-related contexts are still unknown. Here, we investigated the modulation of EC by ATL-1, in a model that mimics tumor extravasation. We observed that the analog inhibited endothelial permeability induced by VEGF, through the stabilization of VE-cadherin/ß-catenin-dependent adherens junctions. We tested the ability of MV3 cells, a highly metastatic melanoma cell line, to transmigrate across unchallenged EC monolayers for 18 h, as compared to NGM normal melanocytes. ATL-1 was able to inhibit only melanoma extravasation. MV3 cells secrete large amounts of VEGF and we observed that ATL-1 per se did not alter this ability. Melanoma cells skills to crossing endothelial monolayers were due to the steady accumulation of tumor-derived VEGF. When endothelial cells were challenged with exogenous VEGF, added at levels comparable to those secreted by MV3 cells over 18 h, and a short-term (4h) transendothelial migration assay was performed, both melanoma and melanocyte cells were able to extravasate, and ATL-1 was able to block the passage of both cells. These results indicate that ATL-1 has a potent inhibitory effect on the permeability induced by VEGF, and that this pharmacological effect could be used to block tumor extravasation across endothelial barriers, with a possible prospect of reducing the haematogenic spread of cancer cells.

Effect of memantine on L-DOPA-induced dyskinesia in the 6-OHDA-lesioned rat model of Parkinson's disease.

An increasing body of experimental evidence demonstrates that the glutamatergic system is involved in the genesis of l-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID). Indeed, the N-methyl-d-aspartate (NMDA) receptor antagonist amantadine is the only anti-dyskinetic compound used in patients, albeit with limited efficacy and side effects. In this study, we investigated the anti-dyskinetic properties of memantine, a non-competitive NMDA receptor antagonist in clinical use for the treatment of dementia, in the 6-hydroxy-dopamine (6-OHDA)-lesion rat model of Parkinson's disease. For comparison, parallel experiments were also performed with amantadine. First, we investigated the acute effect of different doses of memantine (5, 10, 15 and 20mg/kg), and amantadine (10, 20, 40, 60mg/kg) on established dyskinesia induced by L-DOPA (6mg/kg plus benserazide). Results showed that both memantine and amantadine produced a significant reduction of LID. Afterward, drug-naïve and L-DOPA-primed 6-OHDA-lesioned rats were sub-chronically treated with daily injections of L-DOPA (6mg/kg plus benserazide) alone, or in combination with the effective doses of memantine, while amantadine was tested in already dyskinetic rats. Results showed that memantine significantly dampened dyskinesia in both drug-naïve and L-DOPA-primed rats, but only during the first few days of administration. In fact, the anti-dyskinetic effect of memantine was completely lost already at the fifth administration, indicating a rapid induction of tolerance. Interestingly, a 3-week washout period was not sufficient to restore the anti-dyskinetic effect of the drug. Similarly, amantadine was able to dampen already established dyskinesia only during the first day of administration. Moreover, memantine partially decreased the therapeutic effect of L-DOPA, as showed by the result of the stepping test. Finally, loss of the anti-dyskinetic effect of memantine was associated to increased synaptic GluN2A/GluN2B ratio at striatal synaptic membranes. Our results are in line with clinical observations suggesting that NMDA receptor blockade may only be transiently effective against LID in PD patients.

[Sclerochoroidal calcification associated with hypovitaminosis D]. / Calcificaciones esclerocoroideas asociadas a hipovitaminosis D.

CASE REPORT: A 69 year-old woman was referred for a routine visit, during which funduscopy revealed white-yellow subretinal lesions in the superotemporal mid-periphery of both eyes. A and B scan ultrasound showed hyperechogenic lesions located at scleral and choroidal level. Computed tomography revealed posterolateral sclerochoroidal calcifications. Metabolic studies showed a severe vitamin D deficiency with no other remarkable findings. DISCUSSION: Sclerochoroidal calcifications are an infrequent finding that occur as a result of calcium deposit at scleral and choroidal level. They have a characteristic clinical picture and are idiopathic in most cases, but may be associated with some systemic diseases, such as calcium and phosphorous metabolic disorders; this fact warrants a thorough metabolic study. We report a case of bilateral sclerochoroidal calcifications associated with severe vitamin D deficiency with no other significant metabolic findings.

Heme modulates smooth muscle cell proliferation and migration via NADPH oxidase: a counter-regulatory role for heme oxygenase system.

Accumulation of vascular smooth muscle cells (VSMC) in response to inflammatory stimuli is a key event in atherogenesis, which commonly occurs in sinuous vessels with turbulent blood flow what leads to hemolysis and consequent free heme accumulation, a known pro-oxidant and pro-inflammatory molecule. In this work, we investigated the effects of free heme on VSMC, and the molecular mechanisms underlying this process. Free heme induces a concentration-dependent migration and proliferation of VSMC which depends on the production of reactive oxygen species (ROS) derived from NADPH oxidase (NADPHox) activity. Additionally, heme activates redox-sensitive proliferation-related signaling routes, such as mitogen activated protein kinase (MAPK) and NF-κB, and induces heme oxygenase-1 (HO-1) expression. NADPHox-dependent proliferative effect of heme seems to be endogenously modulated by HO since the pretreatment of VSMC with HO inhibitors potentiates heme-induced proliferation and, in parallel, increases ROS production. These effects were no longer observed in the presence of heme metabolites, carbon monoxide and biliverdin. The data indicate that VSMC proliferation induced by heme is endogenously modulated by a critical counter-regulatory crosstalk between NADPHox and HO systems.

A role for dorsal and ventral hippocampus in response learning.

The hippocampus and the striatum have been traditionally considered as part of different and independent memory systems despite growing evidence supporting that both brain regions may even compete for behavioral control in particular learning tasks. In this regard, it has been reported that the hippocampus could be necessary for the use of idiothetic cues in several types of spatial learning tasks. Accordingly, the ventral striatum receives strong anatomical projections from the hippocampus, suggesting a participation of both regions in goal-directed behavior. Our work examined the role of the dorsal and ventral hippocampus on a response learning task. Cytochrome c oxidase (C.O.) quantitative histochemistry was used as an index of brain oxidative metabolism. In addition, determination of C.O. subunit I levels in the hippocampus by western blot analysis was performed to assess the contribution of this subunit to overall C.O. activity. Increased brain oxidative metabolism was found in most of the studied hippocampal subregions when experimental group was compared with a swim control group. However, no differences were found in the amount of C.O. subunit I expressed in the hippocampus by western blot analysis. Our results support that both the dorsal and ventral hippocampus are associated with the use of response strategies during response learning.

Functional interaction between the dorsal hippocampus and the striatum in visual discrimination learning.

The hippocampus and the striatum have traditionally been considered as part of different and independent memory systems. However, there is evidence that supports a functional interaction between the hippocampus and the dorsal striatum at least in particular learning tasks. Here, we evaluated the functional contribution of both brain regions in a visual discrimination learning task using cytochrome c oxidase (CO) quantitative histochemistry. Compared with other brain metabolic mapping techniques, CO activity reflects steady-state neuronal energy demand. Rats were trained for 6 days in a water T-maze to find a hidden escape platform associated with an intramaze visual cue. A control group of animals swam for an equivalent amount of time compared as the trained group but without any escape platform available. After finishing the behavioral task, CO activity was measured in subdivisions of the dorsal hippocampus and the dorsal striatum in both groups. Results show significantly higher CO activity in the CA1 area and the dentate gyrus of the dorsal hippocampus in the trained rats compared with the control group. In addition, a significant negative functional cross-correlation between area CA1 of the dorsal hippocampus and the anterodorsal striatum was found. Our results support current theories on competitive interaction of different memory systems during visual discrimination learning.

[Antileishmanial activity of six extracts from marine organisms]. / Actividad antileishmanial de seis extractos de organismos marinos.

INTRODUCTION: infections caused by protozoa of the genus Leishmania are a global health problem with a high prevalence in underdeveloped countries. There is no vaccine against this disease at present and the treatment used is poor, so the search for more effective and safe medicines is an urgent need. OBJECTIVE: to assess the in vitro antileishmanial activity of six aqueous and hydroalcohol extracts from marine organisms. METHODS: the activity of six extracts against Leishmania amazonensis promastigots and amastigots as well as their toxicity against peritoneal macrophages in BALB/c mice. RESULTS: in the promastigot assay, the extracts from Bryothamnion Iriquetrum, Bunodosoma granulifera, Halimeda opuntia and Physalia physalis showed growth inhibition at concentrations lower than 100 microg/mL whereas in amastigots, these last two extracts were the most active and least toxic with a selectivity index of 6 and 8 respectively. CONCLUSIONS: taking these results into account, it was considered that the H. opuntia and P. physalis extracts showed a promising activity, so it is suggested that further studies on its in vivo activity be conducted.

Analysis of the cytogenetic response in peripheral blood lymphocytes from breast cancer patients following chemotherapy.

The presence of chromosomal aberrations induced in circulating lymphocytes from breast cancer patients during chemotherapy was analyzed. Ten breast cancer patients undergoing neoadjuvant chemotherapy and ten healthy women (controls) were evaluated. Metaphases were obtained from cultures of peripheral lymphocytes stimulated with phytohemaglutinin and metaphase blockage was achieved with colchicine. One hundred metaphases were analyzed for chromosomal aberrations and 1,000 cells for the mitotic index. No significant differences were observed regarding the frequency of chromosomal aberrations, number of cells with chromosomal aberrations and mitotic index between the controls and patients before chemotherapy. However, after the first chemotherapy cycle, the numbers of chromosomal aberrations and cells with them was greater. After the third cycle, the mitotic index was lower, but the fifth cycle produced an increase in relation to the third and fourth cycles. The results suggest that chemotherapy raises the number of chromosomal aberrations and favors persistence of stable chromosomal abnormalities.

A synthetic analog of 15-epi-lipoxin A4 inhibits human monocyte apoptosis: involvement of ERK-2 and PI3-kinase.

Human monocytes play a central function in several steps of the immune response and the process involved in regulating their survival are critical to population control. Lipoxins are lipid mediators and members of the eicosanoid family that exhibit selective stimulatory but nonphlogistic activities in mononuclear cells. In this study, we investigated the effects of 15-epi-16-(para-fluoro)phenoxy-LXA(4) (ATL-1), a synthetic analog of 15-epi-lipoxin A(4), in human monocytes survival and apoptosis. ATL-1 concentration-dependently increased monocyte survival, as a consequence of cell apoptosis reduction by the analog. Treatment of these cells with PD98059 or LY294002 blocked ATL-1 effects, indicating the involvement of ERK-2 and PI3-K, both pathways associated with cell survival. Confirming the activation of these pathways, we demonstrated an increase in ERK-2 nuclear translocation and Akt phosphorylation. Furthermore, we showed that ATL-1 inhibits Bax translocation to the mitochondria. These results confirm a cytoprotective effect of lipoxins in monocytes and might contribute to the elucidation of the mechanisms associated with the resolution phase of the inflammatory process in different pathophysiological events.

A role for the MAPK/ERK pathway in oligodendroglial differentiation in vitro: stage specific effects on cell branching.

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway is important for both long-term survival and timing of the progression of oligodendrocyte differentiation. Oligodendroglial cells treated with MEK inhibitor were distinguished by using stage specific markers: NG2 proteoglycan, A2B5, 2'3'nucleotide-cyclic 3'phosphodiesterase (CNPase) and myelin basic protein (MBP), and classified according to their morphology into different developmental stages. Treatment significantly increased the number of cells with more immature morphologies and decreased the number of mature cells. Furthermore, it increased the number of rounded cells that could not be classified into any of the oligodendroglial developmental stages. The strongest effects were usually observed shortly after treatment. Rounded cells were CNPase/MBP positive and they were not stained by anti-NG2 or A2B5, indicating that they were mature cells unable either to extend and/or to maintain their processes. These data showed an effect of the MAPK/ERK pathway on oligodendroglial branching, with possible consequences for the formation of the myelin sheath.

Procoagulant properties of human MV3 melanoma cells.

A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i) expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa), and ii) direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.

Procoagulant properties of human MV3 melanoma cells

A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i) expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa), and ii) direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.