José Luis Ventura-Gallegos

TiO 2 nanoparticles induce dysfunction and activation of human endothelial cells

Nanoparticles can reach the blood and cause inflammation, suggesting that nanoparticles-endothelial cells interactions may be pathogenically relevant. We evaluated the effect of titanium dioxide nanoparticles (TiO₂) on proliferation, death, and responses related with inflammatory processes such as monocytic adhesion and expression of adhesion molecules (E- and P-selectins, ICAM-1, VCAM-1, and PECAM-1) and with inflammatory molecules (tissue factor, angiotensin-II, VEGF, and oxidized LDL receptor-1) on human umbilical vein endothelial cells (HUVEC). We also evaluated the production of reactive oxygen species, nitric oxide production, and NF-κB pathway activation. Aggregates of TiO₂ of 300 nm or smaller and individual nanoparticles internalized into HUVEC inhibited proliferation strongly and induced apoptotic and necrotic death starting at 5 μg/cm². Besides, TiO₂ induced activation of HUVEC through an increase in adhesion and in expression of adhesion molecules and other molecules involved with the inflammatory process. These effects were associated with oxidative stress and NF-κB pathway activation. In conclusion, TiO₂ induced HUVEC activation, inhibition of cell proliferation with increased cell death, and oxidative stress.

Oxidative stress and apoptosis are induced in human endothelial cells exposed to urban particulate matter.

Correlations between exposure to particle matter (PM) with an aerodynamic diameter <or= 2.5 or 10microm (PM(2.5) and PM(10), respectively) with cardiovascular effects have been demonstrated recently. Endothelial cells seem to play a relevant role in the responses to PM due to their participation in pro-inflammatory events. In this study we determined the effect of PM(2.5) and PM(10) from Mexico City on human endothelial cells by means of evaluating reactive oxygen species (ROS), nitric oxide (NO), NF-kappaB translocation and cell death. For this purpose we used human umbilical vein endothelial cells (HUVEC) as a model. The production of ROS was determined by the reduction of H(2)DCFDA and NO by Griess reagent. The translocation of NF-kappaB was evaluated by Electrophoretic Mobility Shift Assay (EMSA) and the cellular death by the translocation of phosphatidylserine. TNF-alpha was used as a positive control for endothelial cell activation. PM(2.5) and PM(10) induced the production of ROS (77% and 126% increase, respectively, vs. control) and NO (up to 132% and 233% increase, respectively, vs. control). PM(2.5) and PM(10) also induced the nuclear translocation of NF-kappaB. All these events were associated with apoptosis. In conclusion, the activation of HUVEC induced by PM(2.5) and PM(10) is related with an oxidative stress, suggesting that these particles may participate in the development of cardiovascular and inflammatory diseases.

Locus on Chromosome 6p Linked to Elevated HDL Cholesterol Serum Levels and to Protection Against Premature Atherosclerosis in a Kindred With Familial Hypercholesterolemia

Abstract— Heterozygous familial hypercholesterolemia (FH) is a highly atherogenic genetic disorder leading to premature coronary heart disease (CHD), usually before 60 years of age. We studied an extended multigenerational kindred with FH linked to chromosome 1p32 in which atherosclerotic complications were either delayed or prevented in individuals with elevated HDL cholesterol (HDL-C) levels or hyperalphalipoproteinemia (HA). Premature CHD was observed in FH individuals without HA. The study of this family established that the HA trait in the family also followed an autosomal dominant mode of inheritance with a pattern of segregation independent from FH. We identified a locus on chromosome 6 linked to elevated HDL-C levels (HA) in this family. Haplotype analysis refined the localization to a 7.32-cM interval (73 to 80 cM from pter) flanked by markers D6S1280 and D6S1275. Parametric 2-point and multipoint analyses yielded maximum LOD scores of 3.05 and 3.17, respectively. This finding was confirmed with a nonparametric multipoint score of 3.78 (P =0.0009). We propose that this locus, linked to elevated HDL-C levels, confers protection against premature CHD within an FH context.

NF-κB translocation and endothelial cell activation is potentiated by macrophage-released signals co-secreted with TNF-α and IL-1β

Abstract.Objective and Methods: Over-expression of the immune response can lead to pathological conditions such as septic shock or chronic inflammation. Endothelial cell activation by pro-inflammatory products of activated macrophages plays a key role in these conditions. Here we examine the response of primary human endothelial cells (HUVEC) to conditioned media (CM) obtained from LPS-activated macrophages. We further characterized the translocation of NF-κB in the presence of CM by studying the degradation rate of individual IκB isoforms.Results: We show that, as expected, CM induced NF-κB translocation, as well as adhesion capacity in HUVEC. We further show that this response is critically dependent on TNF-α and IL1β naturally present in the CM. However, both the amplitude of NF-κB translocation and adhesiveness observed with CM were well beyond the saturation levels attained after the sole stimulation with recombinant TNF-α and IL-1β, either separately or together. Our results show that CM induced a faster degradation of the IκB-β and IκB-ɛ isoforms than the recombinant cytokines, leading to an enhanced recruitment of NF-κB activity.Conclusions: The above results suggest that the physiological context of factors co-secreted by LPS-activated macrophages enhances TNF-α mediated endothelial activation.

Pyrimethamine induces oxidative stress in Plasmodium yoelii 17XL-infected mice: a novel immunomodulatory mechanism of action for an old antimalarial drug?

Pyrimethamine is an antimalarial drug that has also been used successfully to treat autoimmune diseases such as lymphoproliferative syndrome. In this work, the effect of pyrimethamine (PYR) on the production of free radicals in malaria-infected mice was studied to better understand the drugs immunomodulatory properties. BALB/c and CBA/Ca mice were infected with Plasmodium yoelii 17XL. Seven days after infection, mice were treated with PYR or vehicle and sacrificed 24h later. Treatment with PYR increased superoxide dismutase and glutathione peroxidase activities in erythrocytes and the liver, augmented the levels of nitric oxide in the serum, and upregulated mRNA levels of superoxide dismutase, glutathione peroxidase, catalase, and iNOS in the spleen. In addition, PYR increased lipoperoxidation and protein carbonylation in infected mice. Our results indicate that P. yoelii 17XL reduces oxidative stress in infected cells, while PYR induces it, which is associated with increased parasite elimination. Thus, it is possible that oxidative stress generated by pyrimethamine is also involved in its immunomodulatory mechanism of action.

A noncanonical NF-κB pathway through the p50 subunit regulates Bcl-2 overexpression during an oxidative-conditioning hormesis response

Cells can respond to damage and stress by activating various repair and survival pathways. One of these responses can be induced by preconditioning the cells with sublethal stress to provoke a prosurvival response that will prevent damage and death, and which is known as hormesis. Bcl-2, an antiapoptotic protein recognized by its antioxidant and prosurvival functions, has been documented to play an important role during oxidative-conditioning hormesis. Using an oxidative-hormetic model, which was previously established in the L929 cell line by subjecting the cells to a mild oxidative stress of 50 μM H₂O₂ for 9 h, we identified two different transductional mechanisms that participate in the regulation of Bcl-2 expression during the hormetic response. These mechanisms converge in activating the nuclear transcription factor NF-κB. Interestingly, the noncanonical p50 subunit of the NF-κB family is apparently the subunit that participates during the oxidative-hormetic response.

Primary cultured astrocytes from old rats are capable to activate the Nrf2 response against MPP+ toxicity after tBHQ pretreatment

Astrocytes are key players for brain physiology, protecting neurons by releasing antioxidant enzymes; however, they are also susceptible to damage by neurotoxins. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a central regulator of the antioxidant response, and therefore, pharmacologic inducers are often used to activate this transcription factor to induce cellular protection. To date, it still remains unknown if cells from aged animals are capable of developing this response. Therefore, the purpose of this work was to determine if cortical astrocytes derived from old rats are able to respond to tertbuthyl-hydroquinene (tBHQ) pretreatment and stimulate the Nrf2-antioxidant response pathway to induce an antioxidant strategy against MPP+ toxicity, one of the most used molecules to model Parkinsons disease. Our results show that, although astrocytes from adult and old rats were more susceptible to MPP+ toxicity than astrocytes from newborn rats, when pretreated with tertbuthyl-hydroquinene, they were able to transactivate Nrf2, increasing antioxidant enzymes and developing cellular protection. These results are discussed in terms of the doses used to create protective responses.

Dehydroepiandrosterone inhibits the activation and dysfunction of endothelial cells induced by high glucose concentration

Dehydroepiandrosterone (DHEA), an adrenal steroid, has a protective role against diabetes; however, its mechanisms of action are unknown. Here, we focus on the effect of DHEA on the activation of endothelial cells induced by a high concentration of glucose. Adhesion on U937 cells, expression of adhesion molecules, production of ROS and NO, expression of eNOS, and translocation of NF-κB were evaluated in human umbilical vein endothelial cells (HUVEC) treated with high concentrations of glucose, DHEA, or both. High concentrations of glucose (>20mM) induced an increase in adhesion, an increment in mainly E-selectin and PECAM-1 expression, as well as in ROS and NO production, eNOS expression, translocation of NF-κB, and degradation of its inhibitor IκB-α. DHEA abolished adhesion and the increase of E-selectin, ICAM-1, VCAM-1, and PECAM-1 induced by glucose. In addition, DHEA completely blocked oxidative stress and decreased translocation of NF-κB and the degradation of IκB-α induced by glucose. These results suggest that DHEA protects against the activation of endothelial cells induced by high concentrations of glucose, indicating that DHEA could be useful in the treatment of hyperglycemia and diabetes.

Bcl-2 sustains hormetic response by inducing Nrf-2 nuclear translocation in L929 mouse fibroblasts

Hormesis is the process whereby exposure to a low dose of a chemical agent induces an adaptive effect on the cell or organism. This response evokes the expression of cytoprotective and antioxidant proteins, allowing pro-oxidants to emerge as important hormetic agents. The antiapoptotic protein Bcl-2 is known to protect cells against death induced by oxidants; it has been suggested that Bcl-2 might also modulate steady-state reactive oxygen species levels. The aim of this work was to find out if Bcl-2 might play a role during the hormetic response and in Nrf-2 activation. We have established a model to study the oxidative conditioning hormesis response (OCH) by conditioning the cell line L929 with 50muM H(2)O(2) for 9h. This condition did not induce oxidative damage nor oxidative imbalance, and OCH cells maintained a 70-80% survival rate after severe H(2)O(2) treatment compared to nonconditioned cells. When cells were pretreated with the Bcl-2 inhibitor HA14-1 or were silenced with Bcl-2-siRNA, both the hormetic effect and the Nrf-2 nuclear translocation previously observed were abrogated. Our results suggest a sequence of causal events related to increase in Bcl-2 expression, induction of Nrf-2 activation, and sustained expression of cytoprotective proteins such as GST and gammaGCS.

Glycine suppresses TNF-alpha-induced activation of NF-κB in differentiated 3T3-L1 adipocytes

Glycine strongly reduces the serum levels of pro-inflammatory cytokines and increases the levels of anti-inflammatory cytokines. Recently, glycine has been shown to decrease the expression and secretion of pro-inflammatory adipokines in monosodium glutamate-induced obese (MSG/Ob) mice. It has been postulated that these effects may be explained by a reduction in nuclear factor kappa B (NF-κB) activation. NF-κB is a transcription factor, which is crucial to the inflammatory response. Hasegawa et al. (2011 and 2012) recently reported a glycine-dependent reduction in NF-κB levels. Here, we have investigated the role of glycine in the regulation of NF-κB in differentiated 3T3-L1 adipocytes. The results revealed that pretreatment with glycine interfered with the activation of NF-κB, which has been shown to be stimulated by tumor necrosis factor-alpha (TNF-α). Glycine alone stimulated NF-κB activation in an unusual way such that the inhibitor κB-β (IκB-β) degradation was more significant than that of the inhibitor κB-α (IκB-α) and led to NF-κB complexes comprised of p50 and p65 subunits; IκB-ε degradation did not affect by glycine. These findings suggest that glycine could be used as an alternative treatment for chronic inflammation, which is a hallmark of obesity and other comorbidities, and is characterized by an elevated production of pro-inflammatory cytokines.