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Abstract. Although thiamine pyrophosphate (TPP) is considered a protective agent for endothelial cells, it is still unknown if this is associated with nitric oxide (NO) synthesis. Our aim was to evaluate the synthesis of NO in endothelial cells incubated with TPP and high glucose concentrations. Endothelial cells from the umbilical cord vein from newborns (n = 20), were incubated with 5, 15 or 30 mmol/L glucose, in absence or presence of 0.625 mg/ml of TPP. Our results showed a significant increase in cell proliferation (> 40%; P 90%; P 7%; P 10%; P

Maitotoxin (MTX) is the most potent marine toxin known to date. It is responsible for a particular human intoxication syndrome called ciguatera fish poisoning (CFP). Several reports indicate that MTX is an activator of non-selective cation channels (NSCC) in different cell types. The molecular identity of these channels is still an unresolved topic, and it has been proposed that the transient receptor potential (TRP) channels are involved in this effect. In Xenopus laevis oocytes, MTX at picomolar (pM) concentrations induces the activation of NSCC with functional and pharmacological properties that resemble the activity of TRP channels. The purpose of this study was to characterize the molecular identity of the TRP channel involved in the MTX response, using the small interference RNA (siRNA) approach and the two-electrode voltage-clamp technique (TEVC). The injection of a specifically designed siRNA to silence the transient receptor potential canonical type 1 (TRPC1) protein expression abolished the MTX response. MTX had no effect on oocytes, even at doses 20-fold higher compared to cells without injection. Total mRNA and protein levels of TRPC1 were notably diminished. The TRPC4 siRNA did not change the MTX effect, even though it was important to note that the protein level was reduced by the silencing of TRPC4. Our results suggest that MTX could be a selective activator of TRPC1 channels in X. laevis oocytes and a useful pharmacological tool for further studies on these TRP channels.

Dehydroepiandrosterone (DHEA), an adrenal hormone, has a protective role against cancer. We previously shown that DHEA inhibits the proliferation and migration of cell lines derived from breast cancer; however, the role of DHEA in others events related with these effects are unknown. We hypothesized that DHEA inhibits the expression of proteins and some events related with cell migration and metastasis. We determined the migration in Boyden chambers, the invasion in matrigel, anchorage-independent growth and the formation of spheroids in 3 cell lines (MCF-7, MDA-MB-231, ZR-75-30) derived from breast cancer exposed to DHEA. The secretion of metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and several pro-inflammatory molecules in the secretome of these cells was also evaluated. DHEA inhibited the migration in transwells and the invasion in matrigel of MCF-7 and MDA-MB-231 cells. Besides, DHEA inhibited the anchorage-independent growth on agar and decreased the size of spheroids, and also reduced the secretion of IL-1α, IL-6, IL-8, and TNF-α in all cell lines. Metalloproteinase-1 (MMP-1) secretion was slightly decreased by DHEA treatment in MDA-MB-231 cells. Our results also showed that inhibition of migration and invasion induced by DHEA in breast cancer cells is correlated with the decrease of cytokine/chemokine secretion and the diminution of tumor cells growth. MCF-7 cells were the most responsive to the exposure to DHEA, whereas ZR-75-30 cells responded less to this hormone, suggesting that DHEA could be used in the treatment of breast cancer in early stages.

Obesity, a risk factor for insulin resistance, contributes to the development of type 2 diabetes and cardiovascular diseases. The relationship between increased levels of free fatty acids in the liver mitochondria, mitochondrial function, and ROS generation in rat model of obesity induced by a high-sucrose diet was not sufficiently established. We determined how the bioenergetic functions and ROS generation of the mitochondria respond to a hyperlipidemic environment. Mitochondria from sucrose-fed rats generated H2O2at a higher rate than the control mitochondria. Adding fatty acid-free bovine serum albumin to mitochondria from sucrose-fed rats significantly reduced the rate of H2O2generation. In contrast, adding exogenous oleic or linoleic acid exacerbated the rate of H2O2generation in both sucrose-fed and control mitochondria, and the mitochondria from sucrose-fed rats were more sensitive than the control mitochondria. The increased rate of H2O2generation in sucrose-fed mitochondria corresponded to decreased levels of reduced GSH and vitamin E and increased levels of Cu/Zn-SOD in the intermembrane space. There was no difference between the levels of lipid peroxidation and protein carbonylation in the two types of mitochondria. In addition to the normal activity of Mn-SOD, GPX and catalase detected an increased activity of complex II, and upregulation of UCP2 was observed in mitochondria from sucrose-fed rats, all of which may accelerate respiration rates and reduce generation of ROS. In turn, these effects may protect the mitochondria of sucrose-fed rats from oxidative stress and preserve their function and integrity. However, in whole liver these adaptive mechanisms of the mitochondria were inefficient at counteracting redox imbalances and inhibiting oxidative stress outside of the mitochondria.

A family history of type 2 diabetes mellitus (DM) increases the probability to develop DM and endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) isolated from healthy newborns with familiar background of diverse diseases show early alterations such as less resistance to shear stress. The aim of this study was to evaluate the apoptosis by flow cytometry in HUVECs obtained from healthy newborns with (experimental) and without (control) a strong family history of DM, exposed to different glucose concentrations. Methods HUVECs were incubated in M-199 culture media (containing a 5 mmol/L physiological glucose concentration) or supraphysiological glucose concentrations (15 or 30 mmol/L), for 48 h. Apoptosis was quantified by flow cytometry with annexin V and a polycaspase assay kit (FLICA, Immunochemistry Technologies LLC), and cell death was measurement by propidium iodide (PI, St Cruz Biotechnology Inc) positive stain. Results Experimental HUVECs showed higher levels of apoptosis in the presence of increasing glucose concentration ( p 0.01), whereas control HUVECs showed low levels of apoptosis. Conclusions Our results suggest that HUVECs, isolated from healthy newborns with a strong family history of DM, have an abnormal predisposition to apoptosis.

Background A deficient synthesis of nitric oxide (NO) may play a role in the early endothelial dysfunction of healthy humans with a strong family history of type 2 diabetes (DM2). In this study, we evaluate the intracellular synthesis of NO and the expression of eNOS transcripts in human umbilical vein endothelial cells (HUVECs), exposed to high glucose concentrations, of healthy newborns with (experimental) and without (control) a strong family history of DM2. Methods HUVECs were incubated in M-199 culture media (containing a 5 mmol/L physiological glucose concentration) or supraphysiological glucose concentrations (15 or 30 mmol/L), for 48 h. Flow cytometry, reactive of Griess and RT-PCR were used to determine intracellular NO synthesis, presence of NO metabolites, and expression of eNOS, GLUT1 or p53 transcripts. Results NO synthesis in experimental HUVECs showed a progressive reduction in the presence of increasing glucose concentration (11% for 5 mmol to 8% for 30 mmol; p < 0.01), whereas control HUVECs showed an increase in NO synthesis (3% for 5 mmol to 31% for 30 mmol; p < 0.001). In experimental HUVECs, we found a diminished expression of eNOS and p53, and also an enhanced expression of GLUT1 mRNA transcripts. Control HUVECs showed an increase in eNOS, and no modifications in p53 or GLUT1 mRNA transcripts. Conclusions Our results show how HUVECs, isolated from healthy newborns with a strong family history of DM2, have an abnormal intracellular synthesis of NO and an impaired expression of eNOS, GLUT1 and p53 genes, all associated with NO synthesis. Copyright © 2007 John Wiley & Sons, Ltd.

Bax, a pro-apoptotic member of the Bcl-2 family of proteins has the ability to form transmembrane pores large enough to allow cytochrome c (Cyt c) release, as well as to activate the mitochondrial permeability transition pore (mPTP); however, no differential study has been conducted to clarify which one of these mechanisms predominates over the other in the same system. In the present study, we treated isolated mitochondria from MCF7 cells with recombinant protein Bax and tested the efficacy of the mPTP inhibitor cyclosporin A (CsA) and of the Bax channel blocker (Bcb) to inhibit cytochrome c release. We also, induced apoptosis in MCF7 cell cultures with TNF-α plus cycloheximide to determine the effect of such compounds in apoptosis induction via mPTP or Bax oligomerization. Cytochrome c release was totally prevented by CsA and partially by Bcb when apoptosis was induced with recombinant Bax in isolated mitochondria from MCF7 cells. CsA increased the number of living cells in cell culture, as compared with the effect of Bax channel blocker. These results indicate that mPTP activation is the predominant pathway for Bax-induced cytochrome c release from MCF7 mitochondria and for apoptosis induction in the whole cell.

Objective Women under hormone replacement therapy carry an increased risk of venous thromboembolism (VTE), mostly during the first year. Despite great efforts devoted to hormone therapy research, VTE remains a major drawback of estrogenic therapy, and the search for new compounds continues. We have synthesized and evaluated prolame, an aminoestrogen with anticoagulant properties. The aim of our work was to elucidate the anticoagulant mechanism of prolame. Methods We studied the effects of prolame on nitric oxide (NO) synthesis in cultured endothelial cells and platelets using flow cytometry, on NO metabolites using a modified Griess method, on NO formation in vivo using electron paramagnetic resonance spectroscopy, on participation of nuclear estrogen receptors using flow cytometry, and on endothelial NO synthase (eNOS) mRNA expression using RT-PCR. We also studied the impact of prolame-treated endothelial cells (EC) on ADP-induced platelet aggregation, as well as the ability to prevent occlusive thrombi in an in vivo mice thrombosis model. Results (a) Prolame induces NO production in ECs, platelets, and in a mouse model in vivo. (b) The NO-elevating effect of prolame can only be partially attributed to the nuclear estrogen receptors (ERs) since endothelial nitric oxide synthase (e-NOS) is slightly induced (37%) in ECs treated with prolame. (c) Platelets become 60% less responsive to aggregation induced by 10 μM ADP when in suspension with prolame-treated ECs. (d) Prolame reduces the formation of thrombi in an in vivo thrombosis model. Conclusions Prolame could be a preferred alternative to other estrogens because of its reduced thromboembolic risk.

Statins have antiproliferative and anti-tumoral effects in MCF-7 cells. We determined the effect of statins upon MCF-7 cell cycle, toxicity, cell death, reactive oxygen species (ROS) production and mitochondrial membrane potential. Fluvastatin, simvastatin and atorvastatin inhibited cell proliferation. Antiproliferation was associated with a decrease in the DNA synthesis and a cell cycle arrest in the G1 and G2/M phases. A loss in the mitochondrial membrane potential was observed with fluvastatin. Statins induced increase in ROS production that was associated with cell death, which was abrogated by the antioxidant NAC. Our results suggest that the cytotoxic effect observed is mediated by an oxidative stress.

Background A family history of type 2 diabetes mellitus (DM) increases the probability to develop DM and endothelial dysfunction. The probable mechanism involves augmented reactive oxygen species (ROS) synthesis. The aim of this study was to evaluate the synthesis of ROS in human umbilical vein endothelial cells (HUVECs) obtained from healthy newborns with (experimental) and without (control) a strong family history of type 2 DM, exposed to different glucose concentrations. Methods HUVECs were exposed to various glucose concentrations for 24 and 48 h periods, before cell proliferation, mitochondrial activity, and mitochondrial membrane potential were determined. Intracellular ROS synthesis in the presence or absence of the mitochondrial uncoupler CCCP, cytochalasin B, or diphenyleneiodonium (DPI) was also evaluated. Results As opposed to control HUVECs, we found that experimental HUVECs exposed to 30 mmol/L glucose showed a 50% decrease in cell proliferation, a 90% reduction in mitochondrial activity, and a statistically significant inhibition of ROS synthesis in the presence of CCCP or cytochalasin B; DPI had no effect. Conclusions Our results suggest that mitochondria and NAD(P)H-oxidase from HUVECs obtained from healthy newborns with a family history of DM have an innate deficient response to high glucose concentrations. Copyright © 2006 John Wiley & Sons, Ltd.

Dehydroepiandrosterone (DHEA) has a protective role against atherosclerosis. We determined the effect of pharmacological doses of DHEA upon the adhesion of monocytic U937 cells to human umbilical vein endothelial cells (HUVEC), as well as the expression of adhesion and chemoattractant molecules, the translocation of NF-kappaB, the degradation of IkappaB-alpha and the production of reactive oxygen species (ROS) in HUVEC. Adhesion of U937 cells to DHEA-treated HUVEC was evaluated by co-culture experiments using [(3)H]-thymidine-labeled U937 cells. The expression of adhesion and chemoattractant molecules was evaluated by flow cytometry and RT-PCR, respectively; NF-kappaB translocation was determined by Electrophoretic Mobility Shift Assay (EMSA) and IkappaB-alpha degradation by Western blot. ROS production was determined by the reduction of fluorescent DCFDA. TNF-alpha was used to induce inflammatory responses in HUVEC. One hundred micromolar of DHEA-treatment inhibited the TNF-alpha-induced expression of ICAM-1, E-selectin, ROS production and U937 cells adhesion to HUVEC, and interfered with NF-kappaB translocation and IkappaB-alpha degradation. DHEA at the above mention concentration also inhibited the mRNA expression of MCP-1 and IL-8 in basal conditions but not in TNF-alpha-stimulated conditions. Our results suggest that DHEA inhibits the expression of molecules involved in the inflammatory process, therefore it could be used as an alternative in the treatment of chronic inflammatory diseases such as atherosclerosis.

Dehydroepiandrosterone (DHEA), a steroid hormone, modified the proliferation of human umbilical vein endothelial cells in a dose-dependent manner. Its inactive sulfate ester (DHEA-S) and two of its metabolites -- estradiol and testosterone -- had no inhibitory effect at physiological concentrations. Antiproliferation was associated with arrest in the G1 phase of the cell cycle, but not with cell death, as evaluated by cleavage of poly(ADP-ribose) polymerase and exposure of phosphatidylserine. The effect was not blocked by inhibitors of androgen or estrogen receptors. DHEA diminished the levels of phosphorylated retinoblastoma protein and increased the expression of p53 and p21 mRNAs. These results show that DHEA inhibits endothelial cell proliferation by regulating cell cycle relevant proteins through a cytoplasmic steroid hormone-independent pathway.