Cytoprotection of human endothelial cells from oxidant stress with CDDO derivatives: network analysis of genes responsible for cytoprotection.

AIM: To identify drugs that may reduce the impact of oxidant stress on cell viability. METHODS: Human umbilical vein endothelial cells were treated with 200 nmol/l CDDO-Im (imidazole) and CDDO-Me (methyl) after exposure to menadione and compared to vehicle-treated cells. Cell viability and cytotoxicity were assessed, and gene expression profiling was performed. RESULTS: CDDO-Im was significantly more cytoprotective and less cytotoxic (p < 0.001) than CDDO-Me. Although both provided cytoprotection by induction of gene transcription, CDDO-Im induced more genes. In addition to a higher induction of the key cytoprotective gene heme oxygenase-1 (38.9-fold increase for CDDO-Im and 26.5-fold increase for CDDO-Me), CDDO-Im also induced greater expression of heat shock proteins (5.5-fold increase compared to 2.8-fold for CDDO-Me). CONCLUSIONS: Both compounds showed good induction of heme oxygenase, which largely accounted for their cytoprotective effect. Differences were detected in cytotoxicity at higher doses, indicating that CDDO-Me was more cytotoxic than CDDO-Im. Significant differences were detected in the ability of CDDO-Im and CDDO-Me to affect differential gene transcription. CDDO-Im induced more genes than did CDDO-Me. The source of the differences in gene expression patterns between CDDO-Im and CDDO-Me was not determined but may be important in long-term use of this class of drugs.

Development and validation of an LCMS method to determine the pharmacokinetic profiles of caffeic acid phenethyl amide and caffeic acid phenethyl ester in male Sprague-Dawley rats.

A validated LCMS method was developed for the quantitative determination of caffeic acid phenethyl amide (CAPA) and caffeic acid phenethyl ester (CAPE) from rat plasma. Separation was achieved using a reverse-phase C12 HPLC column (150 × 2.00 mm, 4 µm) with gradient elution running water (A) and acetonitrile (B). Mass spectrometry was performed with electrospray ionization in negative mode. This method was used to determine the pharmacokinetic profiles of CAPA and CAPE in male Sprague-Dawley rats following intravenous bolus administration of 5, 10 and 20 mg/kg of CAPA and 20 mg/kg of CAPE. The pharmacokinetic analysis suggests the lack of dose proportionality in the dose range of 5-20 mg/kg of CAPA. Total clearance values for CAPA ranged from 45 to 156 mL/min and decreased with increasing dose of CAPA. The volume of distribution for CAPA ranged from 17,750 to 52,420 mL, decreasing with increasing dose. The elimination half-life for CAPA ranged from 243.1 to 295.8 min and no statistically significant differences were observed between dose groups in the range of 5-20 mg/kg (p > 0.05). The elimination half-life for CAPE was found to be 92.26 min.

Stability of caffeic acid phenethyl amide (CAPA) in rat plasma.

A validated C18 reverse-phase HPLC method with UV detection at 320 nm was developed and used for the stability evaluation of caffeic acid phenethyl amide (CAPA) and caffeic acid phenethyl ester (CAPE) in rat plasma. CAPA is the amide derivative of CAPE, a naturally occurring polyphenolic compound that has been found to be active in a variety of biological pathways. CAPA has been shown to protect endothelial cells against hydrogen peroxide-induced oxidative stress to a similar degree to CAPE. CAPE has been reported to be rapidly hydrolyzed in rat plasma via esterase enzymes. CAPA is expected to display a longer half-life than CAPE by avoiding hydrolysis via plasma esterases. The stability of CAPA and CAPE in rat plasma was investigated at three temperatures. The half-lives for CAPA were found to be 41.5, 10 and 0.82 h at 25, 37 and 60 °C, respectively. The half-lives for CAPE were found to be 1.95, 0.35 and 0.13 h at 4, 25 and 37 °C, respectively. The energy of activation was found to be 22.1 kcal/mol for CAPA and 14.1 kcal/mol for CAPE. A more stable compound could potentially extend the beneficial effects of CAPE.

Micro-fabricated perforated polymer devices for long-term drug delivery.

Fabrication techniques have been developed to produce a perforated polymer microtube as a drug delivery device. The technique consists of first forming a silicon platform with trenches and alignment marks to hold the tubes for subsequent processing. Photolithography and reactive ion etching with an inductively coupled plasma source were used to fabricate micro holes on the surface of polyimide tubes. Several materials have been used to form the etching mask, including titanium film deposited by e-beam evaporation and SiO(2) and SiN(x) films deposited by high-density plasma chemical vapor deposition (HDPCVD). Three equidistant holes of 20 µm in diameter were fabricated on polyimide tubes (I.D. = 125 µm). The perforated tubes were loaded with ethinyl estradiol and tested for drug release in phosphate buffered saline (pH = 7.1) at 37°C. Zero order release was observed over a period of 30 days with a potential to be extended to 4 years.

Toxicity of aluminum silicates used in hemostatic dressings toward human umbilical veins endothelial cells, HeLa cells, and RAW267.4 mouse macrophages.

BACKGROUND: Aluminum silicates have been used to control bleeding after severe traumatic injury. QuikClot (QC) was the first such product, and WoundStat (WS) is the most recent. We recently observed that WS caused vascular thrombosis when applied to stop bleeding. This study investigated the cellular toxicity of WS in different cell types that may be exposed to this mineral and compared the results with other minerals such as bentonite, kaolin, and QuikClot ACS+ (QC+). METHODS: Human umbilical vein endothelial cells (HUVEC), HeLa cells, and RAW267.4 mouse macrophage-like cells (RAW) were incubated directly with different concentrations of each mineral for 24 hours. Cell viability was determined metabolically using the AlamarBlue fluorescent technique. In another experiment, minerals were exposed to HUVEC via Transwell inserts with a polycarbonate filter (0.4-µm pore size) to prevent direct contact between cells and minerals for determining whether direct exposure or leaching compounds from minerals cause cytotoxicity. RESULTS: Incubation of HUVEC and RAW cells with 1 to 100 µg/mL of the minerals for 24 hours resulted in differential toxicities. The cytotoxicity of WS was equal to that of bentonite and higher than kaolin and QC+. Neither cell type survived for 24 hours in the presence of 100 µg/mL WS or bentonite. These minerals, however, had little effect on the viability of HeLa cells. In the second HUVEC experiment, a 10 times higher concentration of these compounds placed in Transwell inserts yielded no decrease in cell viability. This result indicates that leaching toxicants or binding of nutrients by the ion-exchange properties of minerals did not cause the toxicity. CONCLUSIONS: Although aluminum silicates seem relatively innocuous to epithelial cells, all produced some toxicity toward endothelial cells and macrophages. WS and bentonite were significantly more toxic than kaolin and zeolite present in QC+, respectively, at equivalent doses. The cytotoxic effect seemed to be caused by the direct contact of the minerals with the cells present in wounds. These data suggest that the future clearance of mineral-based hemostatic agents should require more extensive cytotoxicity testing than the current Food and Drug Administration requirements.

Development and characterization of a scalable microperforated device capable of long-term zero order drug release.

A drug delivery system that consists of microperforated polyimide microtubes was developed and characterized. Two groups of polyimide tubes were used. One set consisted of microtubes (I.D. = 125 microm) with 32.9 +/- 1.7 microm size holes. The second set consisted of larger tubes (I.D. = 1000 microm) with 362-542 microm holes. The number of holes was varied between 1 and 3. The small tubes were loaded with crystal violet (CV) and ethinyl estradiol (EE) and the drug release studies were performed in 0.01 M phosphate buffered saline (PBS) (pH 7.1-7.4) at 37.0 +/- 1.0 degrees C for upto 4 weeks. The large tubes were loaded with CV and the drug release was studied in vitro in PBS and also ex vivo in rabbit's vitreous humor. Linear release rates with R(2) > 0.9900 were obtained for all groups with CV and EE. Release rates of 7.8 +/- 2.5, 16.2 +/- 5.5, and 22.5 +/- 6.0 ng/day for CV and 30.1 +/- 5.8 ng/day for EE were obtained for small tubes. For large tubes, a release rate of 10.8 +/- 4.1, 15.8 +/- 4.8 and 22.1 +/- 6.7 microg/day was observed in vitro in PBS and a release rate of 5.8 +/- 1.8 microg/day was observed ex vivo in vitreous humor.

Synthesis of a series of caffeic acid phenethyl amide (CAPA) fluorinated derivatives: comparison of cytoprotective effects to caffeic acid phenethyl ester (CAPE).

A series of catechol ring-fluorinated derivatives of caffeic acid phenethyl amide (CAPA) were synthesized and screened for cytoprotective activity against H2O2 induced oxidative stress in human umbilical vein endothelial cells (HUVEC). CAPA and three fluorinated analogs were found to be significantly cytoprotective when compared to control, with no significant difference in cytoprotection between caffeic acid phenethyl ester (CAPE) and CAPA.

Pharmacokinetics of caffeic acid phenethyl ester and its catechol-ring fluorinated derivative following intravenous administration to rats.

The pharmacokinetic profiles of caffeic acid phenethyl ester (CAPE) and its catechol-ring fluorinated derivative (FCAPE) were determined in rats after intravenous administration of 5, 10 or 20 mg/kg for CAPE and 20 mg/kg for FCAPE, respectively. The plasma concentrations of CAPE and FCAPE were measured using a validated liquid chromatography tandem mass spectrometric method. The pharmacokinetic parameters were estimated using non compartmental analysis (NCA) and biexponential fit. The results showed that the area under the plasma concentration-time curve for CAPE treatment increased in a proportion greater than the increase in dose from 5 to 20 mg/kg of CAPE. Total body clearance values for CAPE ranged from 42.1 to 172 ml/min/kg (NCA) and decreased with the increasing dose of CAPE. Similarly, the volume of distribution values for CAPE ranged from 1555 to 5209 ml/kg, decreasing with increasing dose. The elimination half-life for CAPE ranged from 21.2 to 26.7 min and was independent of dose. That FCAPE was distributed extensively into rat tissues and eliminated rapidly was indicated by a high value of volume of distribution and similar short elimination half-life as that of CAPE.

Cytoprotection of human endothelial cells from menadione cytotoxicity by caffeic acid phenethyl ester: the role of heme oxygenase-1.

Caffeic acid phenethyl ester (CAPE), derived from various plant sources, has been shown to ameliorate ischemia/reperfusion injury in vivo, and this has been attributed to its ability to reduce oxidative stress. Here we investigated the cytoprotection of CAPE against menadione-induced oxidative stress in human umbilical vein endothelial cells (HUVEC) to evaluate potential gene expression involvement. CAPE exhibited dose-dependent cytoprotection of HUVEC. A gene screen with microarrays was performed to identify the potential cytoprotective gene(s) induced by CAPE. Heme oxygenase-1 (HO-1) was highly upregulated by CAPE and this was confirmed with reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting. Inhibition of HO-1 activity using the HO-1 inhibitor tin protoporphyrin IX (SnPPIX), resulted in loss of cytoprotection. Carbon monoxide, one of HO-1 catabolic products appeared to play a small role in CAPE protection. Caffeic acid, a potential metabolite of CAPE with similar free radical scavenging ability, however, didn't show any cytoprotective effect nor induce HO-1. These findings suggest an important role of HO-1 induction in CAPE cytoprotection against oxidant stress, which may not relate to CAPE structural antioxidant activity nor to its traditional enzymatic activity in decomposing heme but to a yet to be determined activity.

Quantitative determination of fluorinated caffeic acid phenethyl ester derivative from rat blood plasma by liquid chromatography-electrospray ionization tandem mass spectrometry.

The quantitative determination of caffeic acid phenethyl ester (CAPE) and its fluorinated derivative (FCAPE) from rat plasma using ultra-performance liquid chromatography with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) is reported. CAPE and FCAPE were extracted using ethyl acetate in the presence of methyl caffeate (MC) as internal standard. Separation was achieved using a C(18) column (2.1 mm x 50 mm, 1.7 microm) and gradient elution with water and acetonitrile containing 0.2% and 0.1% formic acid, respectively. A non-linear response over a broad concentration range (1-1000 ng/ml, r(2)>0.995 using a quadratic regression model and 1/concentration weighting) was obtained. The inter-day and intra-day variability for CAPE and FCAPE were found to be less than 14.2% and 9.5%, respectively. Data are presented to illustrate the practicality of the method for the pharmacokinetic evaluation of CAPE and FCAPE after intravenous administration to rats.

Geldanamycin inhibits hemorrhage-induced increases in caspase-3 activity: role of inducible nitric oxide synthase.

Hemorrhage has been shown to increase inducible nitric oxide synthase (iNOS) and deplete ATP levels in tissues and geldanamycin limits both processes. Moreover, it is evident that inhibition of iNOS reduces caspase-3 and increases survival. Thus we sought to identify the molecular events responsible for the beneficial effect of geldanamycin. Hemorrhage in mice significantly increased caspase-3 activity and protein while treatment with geldanamycin significantly limited these increases. Similarly, geldanamycin inhibited increases in proteins forming the apoptosome (a complex of caspase-9, cytochrome c, and Apaf-1). Modulation of the expression of iNOS by iNOS gene transfection or siRNA treatment demonstrated that the level of iNOS correlates with caspase-3 activity. Our data indicate that geldanamycin limits caspase-3 expression and protects from organ injury by suppressing iNOS expression and apoptosome formation. Geldanamycin, therefore, may prove useful as an adjuvant in fluids used to treat patients suffering blood loss.

Time Course Expression Analysis of 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole Induction of Cytoprotection in Human Endothelial Cells.

1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), a synthetic derivative of oleanolic acid that exhibits antioxidant and anti-inflammatory activity in several animal and in vitro models, has been shown to be beneficial if given after injury. Although induction of heme oxygenase 1 appears to be a major effector of cytoprotection, the mechanism by which the overall effect is mediated is largely unknown. This study evaluated temporal gene expression profiles to better characterize the early transcriptional events and their relationship to the dynamics of the cytoprotective response in human umbilical vein endothelial cells (HUVEC) to CDDO-Im. Time-course gene expression profiling was performed on HUVEC treated with CDDO-Im for 0.5, 1, 3, 6, and 24 hours. More than 10 000 genes were statistically altered in their expression in at least 1 time point across the time course. Large alterations in immediate-early gene expression were readily detectable within 0.5 hour after administration of CDDO-Im.

Geldanamycin treatment inhibits hemorrhage-induced increases in KLF6 and iNOS expression in unresuscitated mouse organs: role of inducible HSP70.

The aim of this study was to determine whether hemorrhage affects the levels of a variety of stress-related proteins and whether changes can be inhibited by drugs reported to provide protection from ischemia and reperfusion injury. Male Swiss Webster mice were subjected to a 40% hemorrhage without resuscitation. Western blot analysis indicated that c-Jun (an AP-1 protein), Kruppel-like factor 6 (KFL6), and inducible nitric oxide synthase (iNOS) were upregulated sequentially in that order. Pretreatment of mice with geldanamycin (GA) 16 h before hemorrhage effectively inhibited the expression of the proteins KLF6 and iNOS, whereas caffeic acid phenethyl ester did not. GA pretreatment increased inducible heat shock protein (HSP) 70 but not HSP90 in both sham and hemorrhagic tissues. The overexpressed inducible HSP70 formed complexes with KLF6 and iNOS. These results suggest that GA may be therapeutically useful for reducing hemorrhage-induced injury when used as a presurgical treatment or when added to resuscitation fluids.

Cytoprotection of human endothelial cells against oxidative stress by 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im): application of systems biology to understand the mechanism of action.

Cellular damage from oxidative stress, in particular following ischemic injury, occurs during heart attack, stroke, or traumatic injury, and is potentially reducible with appropriate drug treatment. We previously reported that caffeic acid phenethyl ester (CAPE), a plant-derived polyphenolic compound, protected human umbilical vein endothelial cells (HUVEC) from menadione-induced oxidative stress and that this cytoprotective effect was correlated with the capacity to induce heme oxygenase-1 (HMOX1) and its protein product, a phase II cytoprotective enzyme. To further improve this cytoprotective effect, we studied a synthetic triterpenoid, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), which is known as a potent phase II enzyme inducer with antitumor and anti-inflammatory activities, and compared it to CAPE. CDDO-Im at 200nM provided more protection to HUVEC against oxidative stress than 20µM CAPE. We explored the mechanism of CDDO-Im cytoprotection with gene expression profiling and pathway analysis and compared to that of CAPE. In addition to potent up-regulation of HMOX1, heat shock proteins (HSP) were also found to be highly induced by CDDO-Im in HUVEC. Pathway analysis results showed that transcription factor Nrf2-mediated oxidative stress response was among the top canonical pathways commonly activated by both CDDO-Im and CAPE. Compared to CAPE, CDDO-Im up-regulated more HSP and some of them to a much higher extent. In addition, CDDO-Im treatment affected Nrf2 pathway more significantly. These findings may provide an explanation why CDDO-Im is a more potent cytoprotectant than CAPE against oxidative stress in HUVEC.

Evaluation of a perforated drug delivery system in mice for prolonged and constant release of a hydrophilic drug.

A drug delivery system (DDS) consisting of a perforated microtube (polyimide, inside diameter = 1.8 mm, tube length = 20 mm, hole size = 0.15 mm) was characterized in vitro and in vivo for its usefulness for long-term release of hydrophilic drugs at a constant rate. Sodium fluorescein mixed with stearic acid was used as the model drug. The DDS was packed with sodium fluorescein and stearic acid in ratios of 50:50, 40:60, and 25:75, respectively, and in vitro drug release studies were performed in saline. Linear release rates with R (2) > 0.9700 were obtained for all groups. Release rates of 1,077.3 ± 264.6, 342.6 ± 146.4, and 14.4 ± 7.0 µg/day for sodium fluorescein were obtained from the three groups, respectively. After monitoring the in vitro release of fluorescein for 11 days, 7 tubes from the 40:60 group were implanted subcutaneously in each individual mice to study the in vivo release of fluorescein from the tubes by measuring the fluorescein in the urine for 84 days. An initial rapid release during the first 4 days was followed by a near zero order fluorescence from the tubes (R (2) = 0.9870). Following completion of the study, the DDSs were retrieved for histology. Morphological analysis indicated no clinical adverse reaction at the site of device implantation specific to the device. The DDS was found to be biocompatible and capable of long-term constant release of a hydrophilic drug such as sodium fluorescein.

17-DMAG diminishes hemorrhage-induced small intestine injury by elevating Bcl-2 protein and inhibiting iNOS pathway, TNF-α increase, and caspase-3 activation.

BACKGROUND: Hemorrhage increases inducible nitric oxide synthase (iNOS) and depletes ATP levels in various tissues. Previous studies have shown that geldanamycin, an inducer of heat shock protein 70kDa (HSP-70) and inhibitor of iNOS, limits both processes. Reduction in NO production limits lipid peroxidation, apoptosome formation, and caspase-3 activation, thereby increasing cellular survival and reducing the sequelae of hemorrhage. The poor solubility of geldanamycin in aqueous solutions, however, limits its effectiveness as a drug. 17-DMAG is a water-soluble analog of geldanamycin that might have greater therapeutic utility. This study investigated the effectiveness of 17-DMAG at reducing hemorrhagic injury in mouse small intestine. RESULTS: In mice, the hemorrhage-induced iNOS increase correlated with increases in Kruppel-like factor 6 (KLF6) and NF-kB and a decrease in KLF4. As a result, increases in NO production and lipid peroxidation occurred. Moreover, hemorrhage also resulted in decreased Bcl-2 and increased TNF-α, IL-6, and IL-10 concentrations, p53 protein, caspase-3 activation, and cellular ATP depletion. A shortening and widening of villi in the small intestine was also observed. Treatment with 17-DMAG significantly reduced the hemorrhage-induced increases in iNOS protein, jejunal alteration, and TNF-α and IL-10 concentrations, but 17-DMAG did not affect the hemorrhage-induced increases in p53 and IL-6 concentration. 17-DMAG treatment by itself upregulated HSP-70, Bcl-2, and p53. CONCLUSION: Since 17-DMAG is water soluble, bioactive, and not toxic, 17-DMAG may prove useful as a prophylactic drug for hemorrhage.

Structure-activity relationships in the cytoprotective effect of caffeic acid phenethyl ester (CAPE) and fluorinated derivatives: effects on heme oxygenase-1 induction and antioxidant activities.

To determine the relationship between catechol ring modifications and the activity of caffeic acid phenethyl ester (CAPE) as a cytoprotective agent, six catechol ring-fluorinated CAPE derivatives were evaluated for their cytoprotective abilities, as well as for their antioxidant and heme oxygenase-1 (HO-1) inducing capacity in a human umbilical vein endothelial cell (HUVEC) model of oxidant stress. To ascertain the involvement of HO-1 induction in the cytoprotective effects of CAPE analogues, their ability to induce HO-1 at 20microM was determined by reverse transcriptase polymerase chain reaction, western blotting and the use of HO-1 inhibitor tin protoporphyrin IX. There was significant induction of HO-1 by CAPE derivatives. Inhibition of HO-1 enzymatic activity resulted in reduced cytoprotection. Modification of the catechol ring of CAPE by introduction of fluorine at various positions resulted in dramatic changes in cytoprotective activity. The maintenance of at least one hydroxyl group on the CAPE catechol ring and the phenethyl ester portion was required for HO-1 induction. CAPE and its derivatives were screened for their ability to scavenge intracellular reactive oxygen species generated in HUVECs by measuring 5-(and-6)-chlormethyl-2', 7'-dichlorodihydrofluorescein diacetate oxidation. The maintenance of 3, 4-dihydroxyl groups on the catechol ring was required for antioxidant activity, but antioxidant activity did not guarantee cytoprotection. Methylation or replacement of one hydroxyl group on the catechol ring of CAPE, however, provided both pro-oxidant and cytoprotective activities. These results indicate that the induction of HO-1 plays a more important role in the cytoprotective activity of CAPE derivatives than their direct antioxidant activity.

Stability of caffeic acid phenethyl ester and its fluorinated derivative in rat plasma.

The stability of caffeic acid phenethyl ester (CAPE) and its fluorinated derivative (FCAPE) in rat plasma and conditions preventing their degradation are reported. Reverse-phase high-pressure liquid chromatography (HPLC) using taxifolin as an internal standard was applied for the quantitative determination of CAPE and FCAPE in rat plasma extracted with ethyl acetate. The assay was validated over a linear range of 0.25-10 microg/mL plasma (r(2) > 0.9990, n = 3). No endogenous interferences were observed in the chromatographic region of interest. The limits of quantification and detection were set at 0.25 and 0.1 microg/mL, respectively. The precision ranged from 0.7 to 13.7% for CAPE, and from 0.4 to 10.4% for FCAPE. Accuracy ranged from -2.8 to 12.4% for CAPE and from -0.6 to 6.8% for FCAPE. The stability was conducted at 4, 25 and 37 degrees C. First-order kinetics was observed for the degradation of CAPE and FCAPE. The energies of activation of CAPE and FCAPE were found to be 17.9 and 20.1 kcal/mol, respectively. Addition of 0.4% of sodium chloride and pH adjustment to 6 prevented their degradation in rat plasma for 24 h and at least one month at -20 degrees C. This study provides useful information for the future pharmacokinetic study of CAPE and FCAPE in rat.

Cytoprotective effect of caffeic acid phenethyl ester (CAPE) and catechol ring-fluorinated CAPE derivatives against menadione-induced oxidative stress in human endothelial cells.

Caffeic acid phenethyl ester (CAPE), a natural polyphenolic compound with many biological activities, has been shown to be protective against ischemia-reperfusion injury. We have synthesized six new catechol ring-fluorinated CAPE derivatives and evaluated their cytotoxic and cytoprotective effects against menadione-induced cytotoxicity in human umbilical vein endothelial cells. These results provide some insights into the structural basis of CAPE cytoprotection in this assay, which does not appear to be based solely on direct antioxidant properties.

Geldanamycin prevents hemorrhage-induced ATP loss by overexpressing inducible HSP70 and activating pyruvate dehydrogenase.

Hemorrhage in mice results in decreased ATP levels in the jejunum, lung, kidney, heart, and brain but not in liver tissue lysates, albeit at variable levels and time kinetics. The decreased protein expression and activity of pyruvate dehydrogenase (PDH) accounted for the hemorrhage-induced ATP loss. Treatment with geldanamycin (GA; 1 microg/g body wt), a known inducer of heat shock protein (HSP)70, inhibited the hemorrhage-induced ATP loss in the jejunum, lung, heart, kidney, and brain. GA was found to increase PDH protein, preserve PDH enzymatic activity, and inhibit mucosal injury in jejunum tissues. GA-induced HSP70i was found to form complexes with PDH protein. HSP70 gene transfer into intestinal epithelial cells promoted PDH and ATP levels, whereas HSP70 short interfering RNA limited them. We conclude that agents able to increase the expression of HSP70 and PDH may be of value in reducing pathology resulting from hemorrhage-associated ATP loss.