Beatriz González-Flecha

Reactive oxygen species in pulmonary inflammation by ambient particulates.

Exposure to ambient air pollution particles (PM) has been associated with increased cardiopulmonary morbidity and mortality, particularly in individuals with pre-existing disease. Exacerbation of pulmonary inflammation in susceptible people (e.g., asthmatics, COPD patients) appears to be a central mechanism by which PM exert their toxicity. Health effects are seen most consistently with PM with aerodynamic diameter < 2.5 micrometers (PM(2.5)), although 10 micrometers < PM < 2.5 micrometers can also be toxic. Through its metal, semi-quinone, lipopolysaccaride, hydrocarbon, and ultrafine constituents, PM may exert oxidative stress on cells in the lung by presenting or by stimulating the cells to produce reactive oxygen (ROS). In vivo, PM increase cytokine and chemokine release, lung injury, and neutrophil influx. In vitro analysis of PM effects on the critical cellular targets, alveolar macrophages, epithelial cells, and neutrophils, demonstrates PM- and oxidant-dependent responses consistent with in vivo data. These effects have been observed with PM samples collected over years as well as concentrated PM(2.5) (CAPs) collected in real time. Oxidative stress mediated by ROS is an important mechanism of PM-induced lung inflammation.

Cardiac Oxidative Stress and Electrophysiological Changes in Rats Exposed to Concentrated Ambient Particles are Mediated by TRP-Dependent Pulmonary Reflexes

Previous studies suggest that, through the stimulation of pulmonary nervous endings, ambient particles modulate the autonomic tone on the heart leading to cardiac oxidant stress and dysfunction. In this paper we investigated the effect of blockade of vanilloid receptor 1 (Transient Receptor Potential Vanilloid Receptor 1 [TRPV1]) on concentrated ambient particles (CAPs)-induced cardiac oxidative stress and dysfunction in a rat model of inhalation exposure. Capsazepine (CPZ), a selective antagonist of TRPV1, was given ip or as an aerosol immediately before exposure to CAPs. Control and CPZ-treated rats were exposed to filtered air or CAPs aerosols for 5 h using the Harvard Ambient Particle Concentrator (mean PM(2.5) mass concentration: 218 +/- 23 mug/m(3)). At the end of the exposure we measured cardiac oxidative stress (in situ chemiluminescence [CL]), lipid peroxidation (thiobarbituric acid reactive substances [TBARS]), and tissue edema. Cardiac function was monitored throughout the exposure. CPZ (ip or aerosol) decreased CAPs-induced CL, lipid TBARS, and edema in the heart, indicating that blocking TRP receptors, systemically or locally, decreases heart CL. CAPs exposure led to significant decreases in heart rate (CAPs 350 +/- 32 bpm, control: 370 +/- 29), and in the length of the QT, RT, Pdur and Tpe intervals. These changes were observable immediately upon exposure and were maintained throughout the 5 h of CAPs inhalation. Changes in cardiac rhythm and electrocardiogram morphology were prevented by CPZ. These data suggest that current abnormalities in CAPs-exposed rats alter the action potentials leading to changes in conduction velocity and ventricular repolarization, and that triggering of TRPV1-mediated autonomic reflexes in the lung is essential for the observed changes in cardiac rhythms.

Mitogen‐activated protein kinases modulate H2O2‐induced apoptosis in primary rat alveolar epithelial cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (ATII) cell apoptosis remain poorly defined. Here we investigated the role of MAPKs as modulators of oxidant‐mediated ATII cell apoptosis using in vitro models of H2O2‐stress. H2O2, delivered either as a bolus or as a flux, lead to time‐ and concentration‐dependent increases in ATII cells apoptosis. Increased apoptosis in primary rat ATII cells was detected at H2O2 concentrations and production rates in the physiological range (1 μM) and peaked at 100 μM H2O2. Immortalized rat lung epithelial cells (RLE), in contrast, required millimolar concentration of H2O2 for maximal responses. H2O2‐induced apoptosis was preceded by rapid activation of all three classes of mitogen‐activated protein kinases (MAPKs): ERK, JNK, and p38. Specific inhibition of JNK using antisense oligonucleotides and ERK and p38 using PD98059 or SB202190, respectively, indicated a pro‐apoptotic role for JNK pathway and an anti‐apoptotic role for ERK‐ and p38‐initiated signaling events. Our data show that the balance between the activation of JNK, ERK, and p38 is a critical determinant of cell fate, suggesting that pharmacological interventions on the MAPK pathways may be useful in the treatment of oxidant‐related lung injury.

Lysosomal and mitochondrial pathways in H2O2-induced apoptosis of alveolar type II cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (AT II) cell apoptosis remain poorly defined. Here we investigated the role of lysosomes as modulators of oxidant‐mediated AT II cell apoptosis using an in vitro model of H2O2‐stress. H2O2 stress led to time‐dependent increases in intracellular oxidants, mitochondrial membrane polarization, cytochrome c release, lysosomal rupture, and AT II cells apoptosis. Increased apoptosis was prevented by specific inhibition of the caspase cascade using the broad‐spectrum caspase inhibitor z‐VAD‐fmk or a caspase 3 inhibitor, or by using functional inhibitors for cathepsin D (pepstatin A) or cathepsin B. Inhibition of cathepsin D also prevented mitochondrial permeabilization and cythocrome c release suggesting that lysosomal rupture precedes and is necessary for the activation of the mitochondrial pathway of cell death.

Genetic Responses to Free Radicals: Homeostasis and Gene Control

Abstract: Gene regulation mechanisms have evolved allowing cells to fine‐tune the level of “endogenous” oxidative stress and to cope with increased free radicals from external sources. Levels of H2O2 are tightly controlled in E. coli by OxyR, which is activated by H2O2 to increase scavenging activities and limit H2O2 generation by the respiratory chain. Sub‐micromolar levels of H2O2 are maintained in mammalian tissues, though the regulatory systems that govern this control are unknown. Excess superoxide triggers the soxRS system in E. coli, which is controlled by the oxidant‐sensitive iron‐sulfur centers of the SoxR protein. Nitric oxide activates SoxR by a different modification of the iron‐sulfur centers. The soxRS regulon mobilizes diverse functions to scavenge free radicals and repair oxidative damage in macromolecules, and other mechanisms that exclude many environmental agents from the cell. Mammalian cells also sense and respond to sub‐toxic levels of nitric oxide, activating expression of heme oxygenase 1 through stabilization of its mRNA. These inductions give rise to adaptive resistance to nitric oxide in neuronal and other cell types.

TOR kinase and ran are downstream from PI3K/Akt in H2O2-induced mitosis

Hydrogen peroxide (H2O2) activates signaling cascades essential for cell proliferation via phosphatidylinositol‐3‐kinase (PI3K) and Akt. Here we show that induction of mitogenic signaling by H2O2 activates sequentially PI3K, Akt, mammalian target of rapamycin (mTOR), and Ran protein. Akt activation is followed by signaling through the mTOR kinase and upregulation of Ran in primary type II pneumocytes, a cell type implicated in the development of lung adenocarcinoma. Pretreatment of the cells with wortmannin, a specific inhibitor of PI3K, or rapamycin, a specific inhibitor of mTOR kinase, prevented H2O2‐increased mitosis. H2O2‐induced Akt ser‐473 phosphorylation and upregulation of Ran protein were prevented by wortmannin but not by rapamycin, indicating that PI3K is upstream of Akt and mTOR is downstream from Akt. Overexpression of myr‐Akt or Ran‐wt in type II pneumocytes increased Akt ser‐473 phosphorylation and mitosis in a catalase‐dependent manner, indicating that H2O2 is essential for Akt and Ran signaling. These results indicate that H2O2‐induced mitogenic signaling in primary type II pneumocytes is mediated by PI3K, Akt, mTOR‐kinase, and Ran protein.

Time course and quantitative analysis of the adaptive responses to 85% oxygen in the rat lung and heart

The phenomenon of oxygen tolerance (resistance to 100% O(2) in rats previously exposed to 85% O(2)) constitutes one of the few models of adaptive responses to oxidative stress in mammals. In vitro studies suggest that reactive oxygen species mediate this response. To test this hypothesis in vivo, we followed the time course of oxidative stress, enzyme induction, and edema in the lung, heart and liver of rats exposed to 85% O(2) for 1 to 5 days. Interestingly, not only the lung, but also the heart of rats exposed to 85% O(2) showed increases in the production of O(*-)(2) (aconitase inactivation) early during the exposure. Increases in O(*-)(2) were associated to oxidative stress (increased in situ chemiluminescence) and transient edema in both tissues. Both the lung and heart displayed induction of superoxide dismutase and reversion of the oxidative stress and damage. The adaptive response in the heart was faster and more efficient, suggesting that this tissue is at a more critical risk when exposed to elevated O(2) concentrations.

Cardiac Oxidative Stress and Dysfunction by Fine Concentrated Ambient Particles (CAPs) are Mediated by Angiotensin-II

Inhalation exposure to fine concentrated ambient particles (CAPs) increases cardiac oxidants by mechanisms involving modulation of the sympathovagal tone on the heart. Angiotensin-II is a potent vasoconstrictor and a sympatho-excitatory peptide involved in the regulation of blood pressure. We hypothesized that increases in angiotensin-II after fine particulate matter (PM) exposure could be involved in the development of cardiac oxidative stress. Adult rats were treated with an angiotensin-converting enzyme (ACE) inhibitor (benazepril®), or an angiotensin receptor blocker (ARB; valsartan®) before exposure to fine PM aerosols or filtered air. Exposures were carried out for 5 hours in the chamber of the Harvard fine particle concentrator (fine PM mass concentration: 440 ± 80 μg/m3). At the end of the exposure the animals were tested for in situ chemiluminescence (CL) of the heart, thiobarbituric acid reactive substances (TBARS) and for plasma levels of angiotensin-II. Also, continuous electrocardiogram (ECG) measurements were collected on a subgroup of exposed animals. PM exposure was associated with statistically significant increases in plasma angiotensin concentrations. Pre-treatment with the ACE inhibitor effectively lowered angiotensin concentration, whereas ARB treatment led to increases in angiotensin above the PM-only level. PM exposure also led to significant increases in heart oxidative stress (CL, TBARS), and a shortening of the T-end to T-peak interval on the ECG that were prevented by treatment with both the ACE inhibitor and ARB. These results show that ambient fine particles can increase plasma levels of angiotensin-II and suggest a role of the renin–angiotensin system in the development of particle-related acute cardiac events.

Cardiac and pulmonary oxidative stress in rats exposed to realistic emissions of source aerosols.

In vivo chemiluminescence (CL) is a measure of reactive oxygen species in tissues. CL was used to assess pulmonary and cardiac responses to inhaled aerosols derived from aged emissions of three coal-fired power plants in the USA. Sprague–Dawley rats were exposed to either filtered air or: (1) primary emissions (P); (2) ozone oxidized emissions (PO); (3) oxidized emissions + secondary organic aerosol (SOA) (POS); (4) neutralized oxidized emissions + SOA (PONS); and (5) control scenarios: oxidized emissions + SOA in the absence of primary particles (OS), oxidized emissions alone (O), and SOA alone (S). Immediately after 6 hours of exposure, CL in the lung and heart was measured. Tissues were also assayed for thiobarbituric acid reactive substances (TBARS). Exposure to P or PO aerosols led to no changes compared to filtered air in lung or heart CL at any individual plant or when all data were combined. POS caused significant increases in lung CL and TBARS at only one plant, and not in combined data from all plants; PONS resulted in increased lung CL only when data from all plants were combined. Heart CL was also significantly increased with exposure to POS only when data from all plants were combined. PONS increased heart CL significantly in one plant with TBARS accumulation, but not in combined data. Exposure to O, OS, and S had no CL effects. Univariate analyses of individual measured components of the exposure atmospheres did not identify any component associated with increased CL. These data suggest that coal-fired power plant emissions combined with other atmospheric constituents produce limited pulmonary and cardiac oxidative stress.

Signaling through Cdk2, importin-α and NuMA is required for H2O2-induced mitosis in primary type II pneumocytes

Proliferation of alveolar type II pneumocytes, the multipotent stem cells of the alveoli, has been implicated in the development of lung adenocarcinoma. Hydrogen peroxide (H(2)O(2)), a potent promoter of signaling cascades, can mediate the transmission of many intracellular signals including those involved in cell proliferation. In this study using rat primary type II pneumocytes, we demonstrate that H(2)O(2) significantly increases mitosis through a pathway that includes cyclin-dependent kinase 2 (Cdk2); importin-alpha, a nuclear trafficking regulator; and nuclear mitotic apparatus protein (NuMA), an essential component in mitotic spindle pole formation. Upon H(2)O(2) treatment, Cdk2 is phosphorylated at position thr-160 leading to increases in importin-alpha and NuMA protein levels and resulting in a significant increase of G(2)/M phase in a roscovatine-dependent manner. Type II pneumocytes transfected with NuMA cDNA also show significant increases in G(2)/M phase, NuMA, Cdk2 thr-160 and importin-alpha expression. These effects were prevented by catalase. These results demonstrate that H(2)O(2) orchestrates a complex signaling network regulating S phase entry, nuclear trafficking and spindle pole formation through activation of Cdk2, importin-alpha, and NuMA. This pathway is essential for H(2)O(2)-induced mitosis in type II pneumocytes.