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Biomedical subjects

Hector R Wong

Publications and source records attributed to Hector R Wong.

At least 19 recordsLinked to original sources
PubMed2003

Intracellular delivery of HSP70 using HIV-1 Tat protein transduction domain.

Heat shock protein 70 (HSP70) is an intracellular stress protein that confers cytoprotection to a variety of cellular stressors. Several lines of evidence have suggested that augmentation of the heat shock response by increasing the expression of HSP70 represents a potential therapeutic strategy for the treatment of critically ill patients. The Tat protein of human immunodeficiency virus 1 (HIV-1) has been used previously to deliver functional cargo proteins intracellularly when added exogenously to cultured cells. We generated a Tat-HSP70 fusion protein using recombinant methods and treated HSF -/- cells with either Tat-HSP70 or recombinant HSP70 prior to exposure to hyperoxia or lethal heat shock. We showed that biologically active, exogenous HSP70 can be delivered into cells using the HIV-1 Tat protein, and that the Tat-mediated delivery of HSP70 confers cytoprotection against thermal stress and hyperoxia and may represent a novel approach to augmenting intracellular HSP70 levels.

Animals↗
PubMed2003

Molecular genetics in the pediatric intensive care unit.

Molecular genetics and genomics have become highly relevant tools in the field of pediatric critical care medicine. The most immediate examples involve the use of molecular genetics as routine diagnostic tests (eg, detection of herpes simplex virus by PCR). Perhaps the most exciting and promising examples involve the research efforts based in these fields, which hold the potential to gain novel insight regarding the complex mechanisms involved in critical illness. Ultimately, the generation of more comprehensive and fundamental knowledge can lead to the design of more effective and specific therapeutic strategies.

Critical Care↗
PubMed2003

Nuclear factor-kappaB as a therapeutic target in critical care medicine.

Nuclear factor-kappaB is a transcriptional factor required for the gene expression of many inflammatory mediators. Nuclear factor-kappaB activation requires removal and degradation of its inhibitor kappaB, an event that occurs after phosphorylation of inhibitor kappaB by a complex of inhibitor kappaB kinases. These events allow nuclear factor-kappaB to translocate into the nucleus, where it binds to kappaB elements and initiates transcription. Inappropriate and prolonged activation of nuclear factor-kappaB has been linked to several diseases associated with inflammatory events, including septic shock, acute respiratory distress syndrome, ischemia, and reperfusion injury. Thus, the key role of nuclear factor-kappaB in regulating inflammation makes this factor a therapeutic target for reducing tissue and organ damage. Regulation and control of nuclear factor-kappaB can be achieved by gene modification strategies or by pharmacologic inhibition of the key components of the cascade that leads to nuclear factor-kappaB activation. The purpose of our review is to describe these novel therapeutic approaches and their potential efficacy.

Critical Care↗
PubMed2002

Phosphatase inhibition leads to activation of IkappaB kinase in murine macrophages.

We have been interested in elucidating the role of intracellular phosphatase activity in the regulation of immune cell activation. To this end, we treated RAW 264.7 murine macrophages with the phosphatase inhibitor, calyculin-A. Treatment with calyculin-A led to activation of IkappaB kinase, degradation of IkappaBalpha, and induced nuclear translocation and DNA binding of NF-kappaB. Each of these effects occurred in both a time- and dose-dependent manner. In addition, each of these effects was negatively modulated by prior induction of the heat-shock response. Despite clear activation of the IkappaB kinase/IkappaBalpha/NF-kappaB pathway, however, phosphatase inhibition did not lead to increased expression of NF-kappaB-dependent genes. Thus, intracellular phosphatase activity is a central regulator of the NF-kappaB signal transduction pathway and is negatively modulated by heat shock. Inhibition of intracellular phosphatase activity with calyculin-A is not sufficient to induce NF-kappaB-dependent gene expression, demonstrating the complexity of NF-kappaB regulation in immune cells.

Animals↗
PubMed2002

Heat shock inhibits activation of NF-kappaB in the absence of heat shock factor-1.

The heat shock response is known to inhibit NF-kappaB activation and NF-kappaB-dependent gene expression. Herein we determined if cells lacking heat shock factor-1 (HSF-1), the major transcription factor regulating heat shock protein gene expression, have an altered ability to modulate NF-kappaB activation. Embryonic fibroblasts from HSF-1-null mutant mice (HSF-1-/- cells) had a drastically reduced ability to express heat shock protein-70 in response to heat shock, compared to embryonic fibroblasts from wild-type mice (HSF+/+ cells). There was no difference, however, in the ability of heat shock to inhibit TNFalpha-mediated NF-kappaB activation, IkappaBalpha degradation, IkappaB kinase activation, and macrophage chemotactic protein-1 expression in the HSF-1-/- cells compared to the HSF-1+/+ cells. These data demonstrate that heat shock inhibits activation of the NF-kappaB/IkappaBalpha pathway and NF-kappaB-dependent gene expression in the absence of an intact heat shock response.

Animals↗
PubMed2002

ARDS. The future.

Improving the course and outcome of patients with ARDS presents a considerable challenge. An important component of meeting this challenge is a more comprehensive understanding of the heterogeneous pathophysiology of ARDS and the biologic response of the individual patient. This understanding may be developed through the power of genomics and its related technology. In particular, it will be crucial to characterize the immunophenotypes of individual patients with ARDS. By understanding the immune status of a given patient at a given point in the disease process, physicians can consider manipulating proinflammatory systems more rationally, such as the complement and chemokine cascades, or the anti-inflammatory arm of the immune system. Finally, a more refined molecular and genetic understanding of endogenous cytoprotective molecules and mechanisms, such as the heat shock response and HO-1, may provide further tools in the future armamentarium against ARDS.

Adult↗
PubMed2002

Temporal and mechanistic effects of heat shock on LPS-mediated degradation of IkappaBalpha in macrophages.

Previous studies demonstrated important interactions between the heat shock response and the IkappaBalpha/NF-kappaB pathway when these two pathways are induced sequentially. One such interaction involves the ability of heat shock to inhibit subsequent degradation of IkappaBalpha in response to a proinflammatory signal. Herein we investigated the temporal relationship between recovery from heat shock and inhibition of IkappaBalpha degradation, and the proximal mechanisms by which heat shock inhibits degradation of IkappaBalpha in macrophages. In RAW 264.7 murine macrophages, prior heat shock inhibited LPS-mediated IkappaBalpha degradation up to 4 h after recovery from heat shock, and this effect correlated with inhibition of LPS-mediated activation of NF-kappaB. Beyond these recovery periods, heat shock did not inhibit IkappaBalpha degradation. IkappaB kinase (IKK) assays demonstrated that heat shock inhibited LPS-mediated activation of IKK up to 1 h after recovery from heat shock. Heat shock also increased intracellular phosphatase activity, and inhibition of intracellular phosphatase activity partially reversed the ability of heat shock to inhibit both LPS-mediated degradation of IkappaBalpha and LPS-mediated activation of IKK. These data demonstrate that the ability of heat shock to inhibit degradation of IkappaBalpha is dependent on the recovery period between the heat shock stimulus and the proinflammatory stimulus. The mechanism by which heat shock inhibits degradation of IkappaBalpha involves dual modulation of IKK and intracellular phosphatase activity.

Animals↗
PubMed2002

A green tea-derived polyphenol, epigallocatechin-3-gallate, inhibits IkappaB kinase activation and IL-8 gene expression in respiratory epithelium.

Interleukin-8 (IL-8) is a principle neutrophil chemoattractant and activator in humans. There is interest in developing novel pharmacological inhibitors of IL-8 gene expression as a means for modulating inflammation in disease states such as acute lung injury. Herein we determined the effects of epigallocatechin-3-gallate (EGCG), a green tea-derived polyphenol, on tumor necrosis factor-alpha (TNF-alpha)-mediated expression of the IL-8 gene in A549 cells. EGCG inhibited TNF-alpha-mediated IL-8 gene expression in a dose response manner, as measured by ELISA and Northern blot analysis. This effect appears to primarily involve inhibition of IL-8 transcription because EGCG inhibited TNF-alpha-mediated activation of the IL-8 promoter in cells transiently transfected with an IL-8 promoter-luciferase reporter plasmid. In addition, EGCG inhibited TNF-alpha-mediated activation of IkappaB kinase and subsequent activation of the IkappaB alpha/NF-kappaB pathway. We conclude that EGCG is a potent inhibitor of IL-8 gene expression in vitro. The proximal mechanism of this effect involves, in part, inhibition of IkappaB kinase activation.

Antioxidants↗
PubMed2002

Ablation of the heat shock factor-1 increases susceptibility to hyperoxia-mediated cellular injury.

High concentrations of oxygen (hyperoxia) are known to cause cellular injury and death. The heat shock response is a highly conserved cellular defense mechanism that protects cells against various environmental stressors, including hyperoxia. Herein we determined the role of heat shock factor-1 (HSF-1), a major component of the heat shock response, in protecting cells against hyperoxia. Embryonic fibroblasts from HSF-1-null mutant mice (HSF-1 -/- cells) were compared to wild-type embryonic fibroblasts (HSF-1 +/+ cells) following 24 hours' exposure to room air or hyperoxia (95% O(2)). Acute survival in hyperoxia was decreased in HSF-1 -/- cells as compared to HSF-1 +/+ cells. Intracellular ATP levels were significantly lower in the HSF-1 -/- cells as compared to the HSF-1 +/+ cells exposed to hyperoxia. Isoprostane levels, a marker of membrane lipid peroxidation, were significantly higher in the HSF-1 -/- cells as compared to the HSF-1 +/+ cells exposed to hyperoxia. Restoration of HSF-1 in the HSF-1 -/- cells by stable transfection with a HSF-1 expression plasmid improved survival in hyperoxia when compared to HSF-1 -/- cells stably transfected with the empty expression vector. Hyperoxia increased activation of HSF-1 in HSF-1 +/+ cells and in HSF-1 -/- cells stably transfected with the HSF-1 expression plasmid. These data demonstrate that HSF-1 plays an important role in conferring resistance to hyperoxia in vitro.

Adenosine Triphosphate↗
PubMed2002

Absence of inducible nitric oxide synthase modulates early reperfusion-induced NF-kappaB and AP-1 activation and enhances myocardial damage.

The role of nitric oxide (NO) generated by the inducible NO synthase (iNOS) during myocardial ischemia and reperfusion is not understood. We investigated the role of iNOS during early reperfusion damage induced in genetically deficient iNOS (iNOS-/-) mice and wild-type littermates. In wild-type mice, ischemia (60 min) and reperfusion (60 min) induced an elevation in serum levels of creatine phosphokinase and myocardial injury characterized by the presence of scattered apoptotic myocytes and mild neutrophil infiltration. Northern blot analysis showed increased expression of iNOS, whose activity was markedly elevated after reperfusion. Immunohistochemistry showed staining for nitrotyrosine; Western blot analysis showed elevated expression of heat shock protein 70 (HSP70), a putative cardioprotective mediator. Plasma levels of nitrite and nitrate, tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and IL-10 were also increased. These events were preceded by degradation of inhibitor kappaBalpha (IkappaBalpha), activation of IkappaB kinase complex (IKK) and c-Jun-NH2-terminal kinase (JNK), and subsequently activation of nuclear factor-kappaB (NF-kappaB) and activator protein 1 (AP-1) as early as 15 min after reperfusion. In contrast, iNOS-/- mice experienced 35% mortality after reperfusion. The extensive myocardial injury was associated with marked apoptosis and infiltration of neutrophils whereas expression of HSP70 was less pronounced. Nitrotyrosine formation and plasma levels of nitrite and nitrate were undetectable. TNF-alpha and IL-6 were increased and IL-10 was reduced in earlier stages of reperfusion. Activation of IKK and JNK and binding activity of NF-kappaB and AP-1 were significantly reduced. Thus, we conclude that iNOS plays a beneficial role in modulating the early defensive inflammatory response against reperfusion injury through regulation of signal transduction.

Animals↗
PubMed2002

Inhaled nitric oxide increases endothelin-1 levels: a potential cause of rebound pulmonary hypertension.

OBJECTIVE: Inhaled nitric oxide (iNO) is front-line therapy for pulmonary hypertension after repair of congenital heart disease. However, little clinical data exists regarding the effects of iNO on regulators of pulmonary vascular resistance. An imbalance between primary vasodilators, such as NO, and vasoconstrictors, such as endothelin-1 (ET-1), has been implicated in rebound pulmonary hypertension upon iNO withdrawal. The objective of this study was to determine whether iNO therapy alters plasma ET-1 levels. DESIGN: This is a prospective study involving pediatric and adult patients at risk for pulmonary hypertension. SETTING: Pediatric patients were in the cardiac intensive care unit and adult patients were in a tertiary-care hospital. PATIENTS: Group 1 included children with congenital heart disease requiring iNO for treatment of pulmonary hypertension after cardiopulmonary bypass (n = 15), group 2 was adults receiving iNO (n = 10), and group 3 included children at risk for pulmonary hypertension after bypass that did not require iNO (n = 8). INTERVENTIONS: Dosages of iNO were 2-60 ppm. The duration of therapy ranged from 23 to 188 hrs in group 1 and 29 to 108 hrs in group 2. MEASUREMENTS AND MAIN RESULTS: Arterial blood was obtained for the measurement of ET-1 levels before and during iNO therapy and 24 hrs after iNO withdrawal. Group 1 mean ET-1 levels increased to 127% of baseline by 12 hrs of iNO, remained elevated at 48 hrs (p < .05), then decreased to 71% of iNO levels 24 hrs after withdrawal (p < .01). Group 2 ET-1 levels increased to 147%, and 137% of baseline at 12 and 24 hrs of iNO therapy, then fell to 68% of baseline within 24 hrs of discontinuing iNO. ET-1 levels in group 3 decreased after surgery (p < .05). CONCLUSIONS: These data suggest that iNO increased plasma ET-1 levels, which subsequently decreased when iNO was discontinued. Increased circulating ET-1 levels might contribute to rebound pulmonary hypertension upon iNO withdrawal.

Administration, Inhalation↗
PubMed2002

Heat shock inhibits tnf-induced ICAM-1 expression in human endothelial cells via I kappa kinase inhibition.

The pulmonary vascular endothelium plays a critical role in lung inflammation. As a result of proinflammatory cytokine expression, adhesion molecules are upregulated on the surface of the endothelial cells. Adhesion molecules facilitate recruitment of leukocytes and thus, have been targeted for potential anti-inflammatory strategies. Prior induction of the stress response through thermal stimulation, or heat shock, alters proinflammatory gene expression by attenuating NF-kappaB signaling. As intercellular adhesion molecule-(ICAM) 1 expression is, in part, NF-kappaB-dependent, we hypothesized that heat shock would inhibit ICAM-1 expression. Heat shocking endothelial cells resulted in heat shock protein (HSP) expression as measured by HSP-70 induction, and decreased TNF-alpha-induced ICAM-1 expression in a manner that appeared to be transcriptionally mediated. Following heat shock, decreased TNF-alpha-induced NF-kappaB activation was observed and was associated with preservation of IkappaB-alpha and a decrease in phosphorylated IkappaB-alpha that correlated to inhibition of I kappa kinase (IKK) activity. Interestingly, exposing respiratory epithelial cells to heat shock, which results in NF-kappaB inhibition, did not affect TNF-induced ICAM-1 expression. We conclude that heat shock decreases endothelial cell ICAM-1 expression via inhibition of IKK activity.

Cell Membrane↗
PubMed2002

Sesquiterpene lactone parthenolide, an inhibitor of IkappaB kinase complex and nuclear factor-kappaB, exerts beneficial effects in myocardial reperfusion injury.

Sesquiterpene lactones are extracts of common medicinal Asteracae plants used in folk medicine for their anti-inflammatory activity. Recently, in vitro studies have shown that these compounds may interfere with pro-inflammatory gene regulation. This study examines the effects of parthenolide, a sesquiterpene lactone, in experimental myocardial ischemia and reperfusion. Myocardial injury was induced in rats by 30 min occlusion and 120 min reperfusion of the left coronary artery. Parthenolide (250 or 500 microg/kg) or vehicle (0.05% Tween 80, 1 mL/kg) was administered intraperitoneally 10 min before reperfusion. In vehicle-treated rats, ischemia and reperfusion caused myocardial injury, as evaluated by infarct size, serum levels of creatine phosphokinase and by histological examination. Elevated tissue levels of myeloperoxidase activity were indicative of a significant infiltration of neutrophils. This event paralleled the occurrence of oxidative damage, as evaluated by a marked increase in tissue malondialdehyde levels. These inflammatory events were preceded by activation of the IkappaB kinase complex (IKK) and partial disappearance of inhibitor-kappaBalpha (IkappaBalpha) in the cytosol and translocation of the nuclear factor-kappaB (NF-kappaB) to the nucleus, as early as 15 min after reperfusion. Administration of parthenolide ameliorated myocardial injury, lowered serum creatine phosphokinase activity, and reduced neutrophil infiltration and the subsequent oxidative damage. These beneficial effects were associated with inhibition of IKK activity, enhanced stability of IkappaBalpha, and inhibition of nuclear translocation of NF-kappaB. The results of this study suggest that parthenolide may be beneficial for the treatment of reperfusion-induced myocardial damage by inhibition of the IKK/NF-kappaB pathway.

Animals↗
PubMed2002

Hyperoxia prolongs tumor necrosis factor-alpha-mediated activation of NF-kappaB: role of IkappaB kinase.

Hyperoxia and tumor necrosis factor-alpha (TNFalpha) are two canonical signals centrally involved in the pathophysiology of acute lung injury. We have attempted to elucidate the effects of these two stimuli on the signal transduction pathways of lung parenchymal cells. In cultured human lung epithelial cells, exposure to hyperoxia alone (95% oxygen) did not affect NF-kappaB activation or degradation of the NF-kappaB inhibitory protein, IkappaB alpha. Stimulation with TNFalpha alone increased NF-kappaB activation within 1 h and induced IkappaB alpha degradation within 0.5 h. After TNFalpha alone, NF-kappaB activation returned to baseline within 2 h and this corresponded with near complete IkappaB alpha resynthesis within 1 h of stimulation. In contrast, simultaneous exposure to hyperoxia and TNFalpha prolonged NF-kappaB activation up to 4 h, and IkappaB alpha degradation up to 2 h after stimulation. Hyperoxia did not affect TNFalpha-mediated resynthesis of IkappaB alpha mRNA. Hyperoxia alone did not induce IkappaB kinase (IKK) activity, but significantly prolonged TNFalpha-mediated activation of IKK activity. Hyperoxia alone did not activate the intercellular adhesion molecule-1 (ICAM-1) promoter, but augmented TNFalpha-mediated activation of the ICAM-1 promoter. These data demonstrate that while hyperoxia alone does not affect activation of NF-kappaB, hyperoxia prolongs TNFalpha-mediated activation of NF-kappaB. The mechanism of this effect involves, in part, prolonged degradation of IkappaB alpha resulting from prolonged activation of IKK.

Cell Line↗
PubMed2002

The stress response decreases NF-kappaB activation in liver of endotoxemic mice.

Recent studies suggest that the stress (heat shock) response protects cells and tissues from inflammatory and other noxious insults. The transcription factor nuclear factor-kappa B (NF-kappaB), normally sequestered in the cytoplasm by its inhibitory protein IkappaB, regulates many genes involved in the inflammatory response to critical illness. Endotoxemia is associated with increased NF-kappaB activity in liver but the effect of the stress response on endotoxin-induced NF-kappaB activation in the liver is not known. We hypothesized that the stress response inhibits NF-kappaB DNA binding activity in liver during endotoxemia. The stress response was induced in mice by hyperthermia (42 degrees C for 3 min) or sodium arsenite (10 mg/kg) and resulted in increased hepatic heat shock protein-72 levels. After induction of the stress response, mice were injected subcutaneously with endotoxin (12.5 mg/kg) or a corresponding volume of sterile saline. NF-kappaB DNA binding activity in the nuclear fraction of liver tissue increased and cytoplasmic IkappaB-alpha levels decreased after endotoxin injection, with a maximal effect seen at 60 min. The endotoxin-induced increase in NF-kappaB DNA binding activity and decrease in IkappaB-alpha levels were inhibited by prior induction of the stress response. In additional experiments, treatment of mice with sodium arsenite after induction of endotoxemia blunted the increase in NF-kappaB activity, indicating a therapeutic potential of sodium arsenite, in addition to its preventive effect. The present results suggest that the protective effects of the stress response in vivo may, at least in part, be due to inhibited NF-kappaB activation.

Animals↗
PubMed2002

Parthenolide, an inhibitor of the nuclear factor-kappaB pathway, ameliorates cardiovascular derangement and outcome in endotoxic shock in rodents.

Parthenolide is a sesquiterpene lactone used in folk medicine for its anti-inflammatory activity. Recent in vitro studies have shown that this compound inhibits the nuclear factor (NF)-kappaB pathway. This study examines the effect of parthenolide in endotoxic shock in rodents. Endotoxic shock was induced by administration of Escherichia coli endotoxin in rats. Three groups of rats received parthenolide (0.25, 0.5, or 1 mg/kg) 15 min before endotoxin; another group received parthenolide (1 mg/kg) 3 h after endotoxin. In vehicle-treated rats, administration of endotoxin caused severe hypotension, which was associated with a marked hyporeactivity to norepinephrine in ex vivo thoracic aortas. Immunohistochemistry showed positive staining for nitrotyrosine, poly(ADP-ribose) synthetase (PARS) and apoptosis, whereas Northern blot analysis showed increased mRNA expression of inducible nitric-oxide synthase (iNOS) in thoracic aortas. Elevated levels of plasma nitrate/nitrite were also found. Elevated lung levels of myeloperoxidase activity were indicative of infiltration of neutrophils. These inflammatory events were preceded by cytosolic degradation of inhibitor kappaBalpha (IkappaBalpha) and activation of nuclear NF-kappaB in the lung. In vivo pretreatment and post-treatment with parthenolide improved the hemodynamic profile and reduced plasma nitrate/nitrite and lung neutrophil infiltration in a dose-dependent fashion. Vascular hyporeactivity of ex vivo aortas was ameliorated. Treatment with parthenolide also abolished nitrotyrosine formation, PARS expression, and apoptosis and reduced iNOS mRNA content in thoracic aortas. DNA binding of NF-kappaB was inhibited by parthenolide in the lung, whereas degradation of IkappaBalpha was unchanged. In a separate set of experiments, pretreatment or post-treatment with parthenolide significantly improved survival in mice challenged with endotoxin. We conclude that parthenolide exerts beneficial effects during endotoxic shock through inhibition of NF-kappaB.

Animals↗