Enhancement of intracellular glutathione protects endothelial cells against oxidant damage

We studied the role of glutathione in the endothelial cell defense against H2O2 damage. Treatment of endothelial cells with buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, depleted the cells of GSH, while L-2-oxothiazolidine-4-carboxylate, an effective intracellular cysteine delivery agent, markedly enhanced endothelial cell GSH concentration. Depletion of intracellular GSH sensitized the endothelial cells to injury by H2O2 either preformed or generated by the glucose-glucose oxidase system. In contrast, an increase of intracellular GSH protected the cells from H2O2 damage. There was an inverse, linear relationship between the intracellular GSH concentrations and killing of endothelial cells by H2O2. Our results suggest that enhancement of endothelial cell GSH may be an alternative approach toward the prevention of oxidant-induced endothelial damage such as adult respiratory distress syndrome.     

Prohibitin protects against oxidative stress-induced cell injury in cultured neonatal cardiomyocyte

Oxidative stress is one of the main causes of myocardial injury, which is associated with cardiomyocyte death. Mitochondria play a key role in triggering the necrosis and apoptosis pathway of cardiomyocytes under oxidative stress. Although prohibitin (PHB) has been acknowledged as a mitochondrial chaperone, its functions in cardiomyocytes are poorly characterized. The present research was designed to investigate the cardioprotective role of PHB in mitochondria. Oxidative stress can increase the PHB content in mitochondria in a time-dependent manner. Overexpression of PHB in cultured cardiomyocytes by transfection of recombinant adenovirus vector containing PHB sense cDNA resulted in an increase of PHB in mitochondria. Compared with the non-transfection cardiomyocytes, PHB overexpression could protect the mitochondria from oxidative stress-induced injury. The mitochondria-mediated apoptosis pathway was consistently suppressed in PHB-overexpressed cardiomyocytes after hydrogen peroxide (H₂O₂) treatment, including a reduced change in mitochondrial membrane permeability transition and an inhibited release of cytochrome c from mitochondria to cytoplasma. As a result, the oxidative stress-induced cardiomyocyte apoptosis was suppressed. These data indicated that PHB protected the cardiomyocytes from oxidative stress-induced damage, and that increasing PHB content in mitochondria constituted a new therapeutic target for myocardium injury. XiaoHua Liu and Zhe Ren contributed equally to this work. ● Prohibitin is an evolutionarily conserved and ubiquitously expressed protein involved in mitochondrial structure, function, and inheritance whose function in cardiomyocyte is not known. In this study, we found oxidative stress could induce increased expression in cardiomyocytes and mitochondrial translocation of PHB, and PHB can protect against oxidative stress in cultured neonatal cardiomyocyte.     

Cyclophilin D deficiency protects against acetaminophen-induced oxidant stress and liver injury

Acetaminophen (APAP) hepatotoxicity is the main cause of acute liver failure in humans. Although mitochondrial oxidant stress and induction of the mitochondrial permeability transition (MPT) have been implicated in APAP-induced hepatotoxicity, the link between these events is unclear. To investigate this, this study evaluated APAP hepatotoxicity in mice deficient of cyclophilin D, a protein component of the MPT. Treatment of wild type mice with APAP resulted in focal centrilobular necrosis, nuclear DNA fragmentation and formation of reactive oxygen (elevated glutathione disulphide levels) and peroxynitrite (nitrotyrosine immunostaining) in the liver. CypD-deficient (Ppif(-/-)) mice were completely protected against APAP-induced liver injury and DNA fragmentation. Oxidant stress and peroxynitrite formation were blunted but not eliminated in CypD-deficient mice. Thus, mitochondrial oxidative stress and induction of the MPT are critical events in APAP hepatotoxicity in vivo and at least part of the APAP-induced oxidant stress and peroxynitrite formation occurs downstream of the MPT.     

miR-324-5p protects against oxidative stress-induced endothelial progenitor cell injury by targeting Mtfr1

Endothelial progenitor cells (EPCs) belong to bone marrow-derived myeloid progenitor cells that have strong proliferative ability. Dysregulation of miRNAs after acute myocardial infarction (AMI) can result in EPCs injury, thus we hypothesize that correction of miRNA expression may contribute to the tolerance of EPCs against oxidative stress. The peripheral blood of healthy volunteers and patients with ST-segment elevation myocardial infarction (STEMI) was clinically collected. EPCs derived from peripheral blood were transfected by miR-324-5p mimic and simultaneously handled with hydrogen peroxide (H₂O₂) to inducing EPCs injury. At 24 hrs after the H₂O₂ treatment, cell viability, the uptake capacity on DiI-Ac-LDL, and carrying ability on FITC-UEA-l and multiplication capacity were analyzed. The mechanism process was carefully researched by valued the characteristics of the mitochondrion morphology, membrane potential, ATP levels, and the expressing of apoptosis pathways. Small RNA sequencing indicated that the expression level of miR-324-5p in peripheral blood EPCs of patients with STEMI was significantly lower compared with the healthy volunteers. The Mtfr1 has been confirmed as a targeted gene of miR-324-5p through miRTarBase software and western blot. The miR-324-5p mimic units could be contributed for the improvement of viability, the uptake capacity on DiI-Ac-LDL and carrying ability on FITC-UEA-l and multiplication capacity on oxidative stress-injured EPCs. miR-324-5p could suppress mitochondrial fragmentation, promote membrane potential, and ATP levels, as well as protect against oxidative stress-induced EPCs apoptosis. Our results suggested that miR-324-5p protects against oxidative stress-induced EPCs injury by regulating Mtfr1.     

Mitofusin-2 protects against cold stress-induced cell injury in HEK293 cells

Mitochondrial impairment is hypothesized to contribute to cell injury during cold stress. Mitochondria fission and fusion are closely related in the function of the mitochondria, but the precise mechanisms whereby these processes regulate cell injury during cold stress remain to be determined. HEK293 cells were cultured in a cold environment (4.0 ± 0.1 °C) for 2, 4, 8, or 12 h. Western blot analyses showed that these cells expressed decreased fission-related protein Drp1 and increased fusion-related protein Mfn2 at 4 h; meanwhile, electron microscopy analysis revealed large and long mitochondrial morphology within these cells, indicating increased mitochondrial fusion. With silencing of Mfn2 but not of Mfn1 by siRNA promoted cold-stress-induced cell death with decreased ATP production in HEK293 cells. Our results show that increased expression of Mfn2 and mitochondrial fusion are important for mitochondrial function as well as cell survival during cold stress. These findings have important implications for understanding the mechanisms of mitochondrial fusion and fission in cold-stress-induced cell injury.     

Curcumin protects retinal cells from light-and oxidant stress-induced cell death

Age-related macular degeneration (AMD) is a complex disease that has potential involvement of inflammatory and oxidative stress-related pathways in its pathogenesis. In search of effective therapeutic agents, we tested curcumin, a naturally occurring compound with known anti-inflammatory and antioxidative properties, in a rat model of light-induced retinal degeneration (LIRD) and in retina-derived cell lines. We hypothesized that any compound effective against LIRD, which involves significant oxidative stress and inflammation, would be a candidate for further characterization for its potential application in AMD. We observed significant retinal neuroprotection in rats fed diets supplemented with curcumin (0.2% in diet) for 2 weeks. The mechanism of retinal protection from LIRD by curcumin involves inhibition of NF-κB activation and down-regulation of cellular inflammatory genes. When tested on retina-derived cell lines (661W and ARPE-19), pretreatment of curcumin protected these cells from H₂O₂-induced cell death by up-regulating cellular protective enzymes, such as HO-1, thioredoxin. Since, curcumin with its pleiotropic activities can modulate the expression and activation of many cellular regulatory proteins such as NF-κB, AKT, NRF2, and growth factors, which in turn inhibit cellular inflammatory responses and protect cells; we speculate that curcumin would be an effective nutraceutical compound for preventive and augmentative therapy of AMD.     

A novel mechanism of coenzyme Q10 protects against human endothelial cells from oxidative stress-induced injury by modulating NO-related pathways

BackgroundAtherosclerosis is a chronic inflammatory disease of the vessel wall associated with oxidized low-density lipoprotein (oxLDL)-induced apoptosis of endothelial cells. Coenzyme Q10 (CoQ10), a potent antioxidant and a critical intermediate of the electron transport chain, has been reported to inhibit LDL oxidation and thus the progression of atherosclerosis. However, its molecular mechanisms on endothelial cells remain still unclarified.MethodsIn this study, primary human umbilical vein endothelial cell cultures treated with oxLDL were used to explore the protective effects of CoQ10.ResultsOur results showed that CoQ10 attenuated the oxLDL-induced generation of reactive oxygen species and improved the antioxidant capacity. CoQ10 also attenuated the oxLDL-mediated down-regulation of endothelial nitric oxide synthase (eNOS) and up-regulation of inducible nitric oxide synthase (iNOS). In addition, CoQ10 suppressed oxLDL-activated NF-κB and downstream inflammatory mediators, including expression of adhesion molecules, release of proinflammatory cytokines and the adherence of monocytic THP-1 cells. Moreover, CoQ10 attenuated oxLDL-altered proapoptotic responses. The inhibitor of eNOS (l-NIO 10 μM) and iNOS (1400W 10 μM) as well as NO enhancer (SNP 10 μM) were used to clean up the mechanism.ConclusionThese results provide new insight into the possible molecular mechanisms by which CoQ10 protects against atherogenesis by NO-related pathways.     

Baicalein protects against doxorubicin-induced cardiotoxicity by attenuation of mitochondrial oxidant injury and JNK activation

The cardiotoxicity of doxorubicin limits its clinical use in the treatment of a variety of malignancies. Previous studies suggest that doxorubicin-associated cardiotoxicity is mediated by reactive oxygen species (ROS)-induced apoptosis. We therefore investigated if baicalein, a natural antioxidant component of Scutellaria baicalensis, could attenuate ROS generation and cell death induced by doxorubicin. Using an established chick cardiomyocyte model, doxorubicin (10 μM) increased cell death in a concentration- and time-dependent manner. ROS generation was increased in a dose-response fashion and associated with loss of mitochondrial membrane potential. Doxorubicin also augmented DNA fragmentation and increased the phosphorylation of ROS-sensitive pro-apoptotic kinase c-Jun N-terminal kinase (JNK). Adjunct treatment of baicalein (25 μM) and doxorubicin for 24 h significantly reduced both ROS generation (587 ± 89 a.u. vs. 932 a.u. ± 121 a.u., P < 0.01) and cell death (30.6 ± 5.1% vs. 46.8 ± 8.3%, P < 0.01). The dissipated mitochondrial potential and increased DNA fragmentation were also ameliorated. Along with the reduction of ROS and apoptosis, baicalein attenuated phosphorylation of JNK induced by doxorubicin (1.7 ± 0.3 vs. 3.0 ± 0.4-fold, P < 0.05). Co-treatment of cardiomyocytes with doxorubicin and JNK inhibitor SP600125 (10 μM; 24 h) reduced JNK phosphorylation and enhanced cell survival, suggesting that the baicalein protection against doxorubicin cardiotoxicity was mediated by JNK activation. Importantly, concurrent baicalein treatment did not interfere with the anti-proliferative effects of doxorubicin in human breast cancer MCF-7 cells. In conclusion, baicalein adjunct treatment confers anti-apoptotic protection against doxorubicin-induced cardiotoxicity without compromising its anti-cancer efficacy.     

Treatment with outgrowth endothelial cells protects cerebral barrier against ischemic injury

Background and aimsWe have previously reported that outgrowth endothelial cells (OECs) restore cerebral endothelial cell integrity through effective homing to the injury site. This study further investigates whether treatment with OECs can restore blood–brain barrier (BBB) function in settings of ischemia-reperfusion injury both in vitro and in vivo.MethodsAn in vitro model of human BBB was established by co-culture of astrocytes, pericytes, and human brain microvascular endothelial cells (HBMECs) before exposure to oxygen-glucose deprivation alone or followed by reperfusion (OGD±R) in the absence or presence of exogenous OECs. Using a rodent model of middle cerebral artery occlusion (MCAO), we further assessed the therapeutic potential of OECs in vivo.ResultsOwing to their prominent antioxidant, proliferative, and migratory properties, alongside their inherent capacity to incorporate into brain vasculature, treatments with OECs attenuated the extent of OGD±R injury on BBB integrity and function, as ascertained by increases in transendothelial electrical resistance and decreases in paracellular flux across the barrier. Similarly, intravenous delivery of OECs also led to better barrier protection in MCAO rats as evidenced by significant decreases in ipsilateral brain edema volumes on day 3 after treatment. Mechanistic studies subsequently showed that treatment with OECs substantially reduced oxidative stress and apoptosis in HBMECs subjected to ischemic damages.ConclusionThis experimental study shows that OEC-based cell therapy restores BBB integrity in an effective manner by integrating into resident cerebral microvascular network, suppressing oxidative stress and cellular apoptosis.     

Tangeretin protects human brain microvascular endothelial cells against oxygen-glucose deprivation-induced injury

Tangeretin, a citrus flavonoid extracted from the peel of citrus fruits, was reported to possess antiasthmatic, antioxidant, anti-inflammatory, and neuroprotective properties. However, the effect of tangeretin on human brain microvascular endothelial cells (HBMECs) has not been examined. This study was designed to investigate the protective effects of tangeretin on oxygen-glucose deprivation (OGD)-induced injury of HBMECs, and explore the underlying mechanisms. Our results showed that tangeretin improved HBMECs viability in response to OGD. In addition, tangeretin was able to increase the activity of superoxide dismutase and decrease the levels of reactive oxygen species and malondialdehyde (MDA), as well as ameliorate cell apoptosis in OGD-stimulated HBMECs. Mechanistic studies showed that tangeretin prevented the activation of JNK signaling pathway in OGD-stimulated HBMECs. Taken together, our current study demonstrated that tangeretin could ameliorate OGD-induced HBMECs injury through the JNK signaling pathway. Thus, tangeretin might be used as a therapeutic strategy for ischemia-reperfusion brain injury and related diseases.     

The anti-cancer drug, doxorubicin, causes oxidant stress-induced endothelial dysfunction

The anticancer drug doxorubicin (DOX) is toxic to target cells, but also causes endothelial dysfunction and edema, secondary to oxidative stress in the vascular wall. Thus, the mechanism of action of this drug may involve chemotoxicity to both cancer cells and to the endothelium. Indeed, we found that the permeability of monolayers of bovine pulmonary artery endothelial cells (BPAEC) to albumin was increased by approximately 10-fold above control, following 24-h exposure to clinically relevant concentrations of DOX (up to 1 microM). DOX also caused >4-fold increases in lactate dehydrogenase leakage and large decreases in ATP and reduced glutathione (GSH) in BPAECs, which paralleled the increases in endothelial permeability. A large part of the ATP loss could be attributed to DOX-induced hydrogen peroxide production which inhibited key thiol-enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate dehydrogenase (G6PDH). Depletion of reduced nicotinamide adenine dinucleotide phosphate (NADPH) appeared to be a major factor leading to DOX-induced GSH depletion. At low concentrations, the sulfhydryl reagent, iodoacetate (IA), inhibited GAPDH, caused a decrease in ATP and increased permeability, without inhibiting G6PDH or decreasing GSH. These results, coupled with those of previous work on a related quinone, menadione, suggest that depletion of either GSH or ATP may lead independently to endothelial dysfunction during chemotherapy, contributing to the cardiotoxicity and other systemic side-effects of the drug.     

The thioredoxin system protects ribosomes against stress-induced aggregation

We previously showed that thioredoxins are required for dithiothreitol (DTT) tolerance, suggesting they maintain redox homeostasis in response to both oxidative and reductive stress conditions. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to DTT to identify cell functions involved in resistance to reductive stress. We identified 195 mutants, whose gene products are localized throughout the cell. DTT-sensitive mutants were distributed among most major biological processes, but they particularly affected gene expression, metabolism, and the secretory pathway. Strikingly, a mutant lacking TSA1, encoding a peroxiredoxin, showed a similar sensitivity to DTT as a thioredoxin mutant. Epistasis analysis indicated that thioredoxins function upstream of Tsa1 in providing tolerance to DTT. Our data show that the chaperone function of Tsa1, rather than its peroxidase function, is required for this activity. Cells lacking TSA1 were found to accumulate aggregated proteins, and this was exacerbated by exposure to DTT. Analysis of the protein aggregates revealed that they are predominantly composed of ribosomal proteins. Furthermore, aggregation was found to correlate with an inhibition of translation initiation. We propose that Tsa1 normally functions to chaperone misassembled ribosomal proteins, preventing the toxicity that arises from their aggregation.     

Adrenomedullin protects rat cerebral endothelial cells from oxidant damage in vitro

Increased permeability and reduced cerebral endothelial cell (CEC) viability induced by oxidative stress are the hallmarks of the blood-brain barrier disruption. In our experiments hydrogen peroxide (H2O2, 0.5 mM) induced a continuous decrease of the transendothelial electrical resistance (TEER) and resulted in intercellular gap formations in cultured rat CECs. Adrenomedullin (AM) increased TEER, enhanced peripheral localization of F-actin bands and attenuated the increased permeability induced by H2O2. Furthermore, AM treatment preserved mitochondrial membrane potential, attenuated cytochrome c release, and consequently improved CEC viability in H2O2 treated cultures. These results suggest that AM treatment protects CECs against oxidative injury.     

Vitamin B12 protects against superoxide-induced cell injury in human aortic endothelial cells

Superoxide (O₂^•−) is implicated in inflammatory states including arteriosclerosis and ischemia-reperfusion injury. Cobalamin (Cbl) supplementation is beneficial for treating many inflammatory diseases and also provides protection in oxidative-stress-associated pathologies. Reduced Cbl reacts with O₂^•− at rates approaching that of superoxide dismutase (SOD), suggesting a plausible mechanism for its anti-inflammatory properties. Elevated homocysteine (Hcy) is an independent risk factor for cardiovascular disease and endothelial dysfunction. Hcy increases O₂^•− levels in human aortic endothelial cells (HAEC). Here, we explore the protective effects of Cbl in HAEC exposed to various O₂^•− sources, including increased Hcy levels. Hcy increased O₂^•− levels (1.6-fold) in HAEC, concomitant with a 20% reduction in cell viability and a 1.5-fold increase in apoptotic death. Pretreatment of HAEC with physiologically relevant concentrations of cyanocobalamin (CNCbl) (10-50 nM) prevented Hcy-induced increases in O₂^•− and cell death. CNCbl inhibited both Hcy and rotenone-induced mitochondrial O₂^•− production. Similarly, HAEC challenged with paraquat showed a 1.5-fold increase in O₂^•− levels and a 30% decrease in cell viability, both of which were prevented with CNCbl pretreatment. CNCbl also attenuated elevated O₂^•− levels after exposure of cells to a Cu/Zn-SOD inhibitor. Our data suggest that Cbl acts as an efficient intracellular O₂^•− scavenger.     

Expression of uncoupling protein 3 in mitochondria protects against stress-induced myocardial injury: a proteomic study

It has been confirmed that stress plays an important role in the induction and development of cardiovascular diseases, but its mechanism and molecular basis remain unknown. In the present study, a myocardial injury model induced by restraint stress was established in rat. To screen for the related proteins involved in stress-induced myocardial injury, proteomic techniques based on 2-DE and mass spectrometry were used. In our results, ten proteins were found to be altered. The expression of eight of these proteins was increased after restraint stress, including cardiac myosin heavy chain, dihydrolipoamide succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial aldehyde dehydrogenase, H⁺-transporting ATP synthase, albumin, and apolipoprotein A-I precursor. The expression of uncoupling protein 3 (UCP3) and mitochondrial aconitase was decreased. Most of the proteins were related to energy metabolism. Further research indicated that UCP3 may mediate the myocardial cell response induced by restraint stress.     

Inhibition of angiotensin-converting enzyme protects endothelial cell against hypoxia/reoxygenation injury

Cardiovascular tissue injury in ischemia/reperfusion has been shown to be prevented by angiotensin-converting enzyme (ACE) inhibitors. However, the mechanism on endothelial cells has not been assessed in detail. Cultured human aortic endothelial cells (HAEC) were exposed to hypoxia with or without reoxygenation. Hypoxia enhanced apoptosis along with the activation of caspase-3. Reoxygenation increased lactate dehydrogenase release time-dependently, along with an increase of intracellular oxygen radicals. ACE inhibitor quinaprilat and bradykinin significantly lessened apoptosis and lactate dehydrogenase release with these effects being diminished by a kinin B2 receptor antagonist and a nitric oxide synthase inhibitor. In conclusion, hypoxia activated the suicide pathway leading to apoptosis of HAEC by enhancing caspase-3 activity, while subsequent reoxygenation induced necrosis by enhancing oxygen radical production. Quinaprilat could ameliorate both apoptosis and necrosis through the upregulation of constitutive endothelial nitric oxide synthase via an increase of bradykinin, with the resulting increase of nitric oxide.     

Intralysosomal iron chelation protects against oxidative stress-induced cellular damage

Oxidant-induced cell damage may be initiated by peroxidative injury to lysosomal membranes, catalyzed by intralysosomal low mass iron that appears to comprise a major part of cellular redox-active iron. Resulting relocation of lytic enzymes and low mass iron would result in secondary harm to various cellular constituents. In an effort to further clarify this still controversial issue, we tested the protective effects of two potent iron chelators--the hydrophilic desferrioxamine (dfo) and the lipophilic salicylaldehyde isonicotinoyl hydrazone (sih), using cultured lysosome-rich macrophage-like J774 cells as targets. dfo slowly enters cells via endocytosis, while the lipophilic sih rapidly distributes throughout the cell. Following dfo treatment, long-term survival of cells cannot be investigated because dfo by itself, by remaining inside the lysosomal compartment, induces apoptosis that probably is due to iron starvation, while sih has no lasting toxic effects if the exposure time is limited. Following preincubation with 1 mM dfo for 3 h or 10 microM sih for a few minutes, both agents provided strong protection against an ensuing approximately LD50 oxidant challenge by preventing lysosomal rupture, ensuing loss of mitochondrial membrane potential, and apoptotic/necrotic cell death. It appears that once significant lysosomal rupture has occurred, the cell is irreversibly committed to death. The results lend strength to the concept that lysosomal membranes, normally exposed to redox-active iron in high concentrations, are initial targets of oxidant damage and support the idea that chelators selectively targeted to the lysosomal compartment may have therapeutic utility in diminishing oxidant-mediated cell injury.     

Schisandrin B enhances the glutathione redox cycling and protects against oxidant injury in different types of cultured cells

Tert-butylhydroperoxide (tBHP) challenge caused an initial depletion of cellular reduced glutathione (GSH), which was followed by a gradual restoration of cellular GSH in AML12, H9c2, and differentiated PC12 cells. The time-dependent changes in cellular GSH induced by tBHP were monitored as a measure of GSH recovery capacity (GRC), of which glutathione reductase (GR)-mediated glutathione redox cycling and γ-glutamate cysteine ligase (GCL)-mediated GSH synthesis were found to play an essential role. While glutathione redox cycling sustained the GSH level during the initial tBHP-induced depletion, GSH synthesis restores the GSH level thereafter. The effects of (-)schisandrin B [(-)Sch B] and its analogs (Sch A and Sch C) on GRC were also examined in the cells. (-)Sch B and Sch C, but not Sch A, ameliorated the extent of tBHP-induced GSH depletion, indicative of enhanced glutathione redox cycling. However, the degree of restoration of GSH post-tBHP challenge was not affected or even decreased. Pretreatment with (-)Sch B and Sch C, but not Sch A, protected against oxidant injury in the cells. The (-)Sch B afforded cytoprotection was abolished by N,N'-bis(chloroethyl)-N-nitrosourea pretreatment suggesting the enhancement of glutathione redox cycling is crucially involved in the cytoprotection afforded by (-)Sch B against oxidative stress-induced cell injury.     

Activated epidermal growth factor receptor (ErbB1) protects the heart against stress-induced injury in mice

Acute, high-intensity stress induces necrotic lesions in the heart. We found that restraint-and-cold (4 degrees C) exposure (RCE) raises plasma lactate dehydrogenase (LDH), creatine kinase (CK), and transaminase activity in a time-dependent manner, with a peak value 7 h after stimulus cessation. At 24 h, signs of necrotic lesions were observed in paraffin sections stained with hematoxylineosin: focal accumulation of mononuclear cells in subendocardial areas of the left ventricle wall and focal hemorrhage in papillary muscles. In contrast, intermale fighting (IF) did not increase plasma CK activity, although LDH and transaminase activities did increase. In IF, no histological evidence of heart injury was observed. Because IF, but not RCE, increased plasma epidermal growth factor (EGF) concentration by approximately 1,000-fold, we hypothesized that EGF receptor (ErbB1) activation may protect the heart against stress-induced injury. To examine this hypothesis, we injected the ErbB1 tyrosine kinase inhibitor tyrphostin AG-1478 (25 mg/kg ip) immediately before mice were exposed to IF. After 3 h, plasma activities of LDH-1 and CK increased. Plasma enzyme activities were as low in control mice (injected with vehicle alone) as in nonfighting mice. In the last experiment, we injected EGF (0.25 mg/kg ip) 20 min before exposing mice to RCE. After 7 h, plasma LDH-1 and CK activities were significantly lower in these animals than in mice injected with vehicle. The effect required ErbB1 activation, because simultaneous administration of AG-1478 completely abolished the effect of exogenous EGF. We conclude that activated ErbB1, by endogenous or exogenous ligands, may protect the heart against stress-induced injury.