Hemorrhagic shock (HS) results in the disruption of the microvascular endothelial cell barrier leading to vascular hyperpermeability (1-3). The formation of reactive oxygen species (ROS) and the activation of apoptotic signaling have been reported as part of HS (4-7). The activation of the mitochondrion-mediated "intrinsic" apoptotic signaling pathway has been implicated in cell death after I/R injury (6, 8). The intrinsic pathway of apoptosis is mediated through mitochondrial release of cytochrome c, second mitochondrion-derived activator of caspases, and apoptosis-inducing factor, all of which are regulated by proapoptotic and antiapoptotic Bcl-2 family proteins, such as Bax/Bak and Bcl-2/xL (9). Our laboratory has recently identified a positive relationship between apoptotic signaling and vascular hyperpermeability after HS (5). That study demonstrated that activation of intrinsic apoptotic signaling cascade is a key factor that ultimately leads to vascular hyperpermeability after HS. The purpose of this study was to test the effectiveness of α-lipoic acid in controlling vascular hyperpermeability after HS and to determine its involvement in the intrinsic apoptotic signaling pathway.
α-Lipoic acid is a disulfide compound that is a cofactor in vital energy-producing reactions in the body. It is an endogenous antioxidant that neutralizes free radicals in both hydrophobic and hydrophilic regions of cells. Most of the metabolic reactions in which α-lipoic acid participate occur in the mitochondria. It is a constituent of biological membranes and an important cofactor of mitochondrial dehydrogenases. α-Lipoic acid is easily absorbed from the diet, and in mammalian cells, it is readily converted to its reduced from, dihydrolipoic acid (10). It has been shown to prevent oxidant-induced cell death (11, 12) and has been used in the prevention and treatment of a variety of diseases including neurodegenerative diseases, diabetes, radiation injury, and human immunodeficiency virus infection (12). Recently, there was a renewed interest in evaluating its potential for controlling warm ischemia, liver I/R injury, and liver regeneration (13, 14). However, there is no information available on its involvement in vascular hyperpermeability after HS.
Vascular hyperpermeability is mostly dependent upon the integrity of endothelial cell adherens junctions and occurs when cells undergo morphological changes resulting in the uncoupling of these junctions. The various mechanisms that disrupt adherens junctions leading to hyperpermeability after HS are not clearly known. β-Catenin, along with other proteins, plays a significant role in the maintenance of adherens junctions between endothelial cells (15). β-Catenin is a regulator in cadherin-mediated endothelial cell adhesion, and its proteolytic cleavage may result in the detachment of cells leading to vascular hyperpermeability. Proteases of the caspase family play a major role in the maintenance of endothelial adherens junctions, by their ability to cleave catenins. Caspase 3-mediated proteolytic cleavage of catenins has been reported in various cell types (16, 17). Caspases cleave the components of cell-cell (β- and γ-catenin) and cell-matrix (focal adhesion kinase and p130(Cas) adherens junctions during apoptosis with dose and time requirements that paralleled those seen for barrier dysfunction and detachment (15). Mitochondrial release of cytochrome c plays the most facilitatory role among the many factors that regulate caspase activation (9). Mitochondrial release of cytochrome c is a trigger for the release of apoptosome assembly from apoptotic protease activating factor 1, adenosine triphosphate, and procaspase 9, which activates caspase 3 and caspase 7 (9). Translocation of cytochrome c from the mitochondria to the cytosol through mitochondrial transition pores is precisely controlled by the change in mitochondrial transmembrane potential. The mitochondrial transition pores are high-conductance calcium-sensitive channels in the mitochondrial inner membrane of mitochondria that allow the nonselective diffusion of solutes across the membrane (18). Mitochondrial transition pores play a crucial role in apoptotic signaling and cell death in a variety of cells and organ systems (18, 19). Various studies have shown that mitochondrial transition pores can be activated by Ca2+ and reactive oxygen species (ROS) (20, 21). Recent studies from our laboratory have shown that a decrease in mitochondrial transmembrane potential, increased mitochondrial release of cytochrome c, and activation of caspase 3 occur in vascular hyperpermeability after HS (5).
We have hypothesized that α-lipoic acid, an endogenous molecule with antioxidant and antiapoptotic properties, would attenuate vascular hyperpermeability after HS by preventing the activation of apoptotic signaling.
MATERIALS AND METHODS
Male Sprague-Dawley rats (275-325 g) obtained from Charles River Laboratories (Wilmington, Mass) were housed in the institutional animal facility at the Texas A&M University Health Science Center, Scott & White Memorial Hospital. This facility is approved by the American Association for Accreditation of Laboratory Animal Care. All experiments using animals were performed after obtaining approval from the Institutional Animal Care and Use Committee, in accordance with the National Institutes of Health guidelines for laboratory animal care and use in research. The rats were maintained on a 12-h dark/light cycle, with free access to food and water. The room temperature and humidity were maintained at 25°C ± 2°C and 55%, respectively. The rats were fasted for 18 h and given water ad libitum before each experiment.
Chemicals and solutions
α-Lipoic acid was obtained from Sigma (St Louis, Mo). Fluorescein isothiocyanate (FITC)-bovine albumin (FITC-albumin; Sigma) was used as the test solute for the permeability measurements. The test solution was prepared by dissolving the FITC-albumin into saline (50 mg/kg). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide (JC-1; Cell Technology Inc, Mountain View, Calif) was used for measuring mitochondrial transmembrane potential. The JC-1 reagent was prepared by reconstituting the lyophilized reagent with 500 μL of dimethyl sulfoxide to obtain a 100X stock solution. Immediately before the experiments, the 100X solution was diluted 1:100 in 1X assay buffer. The JC-1 reagent was superfused over the exposed mesenteric vessels at a volume of 300 μL into a bath of 2 mL of saline. The JC-1 rapidly diffused into the vasculature (13-15 mL blood volume) and was detected in the microvascular endothelial cells. Dihydrorhodamine 123 was obtained from Sigma.
Animal surgery and intravital microscopy
The rats were anesthetized by a single i.m. injection of 50% urethane (1.5 g/kg). Polyethylene tubing (PE-50; inner diameter, 0.58 mm) was placed in the right internal jugular vein to give fluid (isotonic sodium chloride solution, 2 mL/h) continuously by an infusion pump (Harvard Apparatus, South Natick, Mass) and in the right carotid artery for blood withdrawal. The MAP was monitored continuously using a PE-50 cannula placed in the left femoral artery connected to a blood pressure analyzer (Dig-Med, BPA 400A, Micromed, Louisville, Ky). A midline laparotomy incision was performed to expose a section of mesentery, from the proximal ileum, for exteriorization. The rats were placed on a temperature-controlled Plexiglas platform mounted to an intravital upright microscope (Nikon E600, Tokyo, Japan), in the lateral decubitus position. The mesentery was draped over a Plexiglas stage and maintained at 37°C. The mesentery was superfused with isotonic sodium chloride solution at 2 mL/min and covered with plastic wrap to reduce evaporation. Postcapillary venules with diameters of 20 to 35 μm were selected for study with a Nikon 20× objective, 0.45 to 2.16 mm working distance (Nikon Instruments, Inc, Natick, Mass). Images were obtained with a Photometric Cascade Camera (Roper Scientific, Tucson, Ariz). A video time and date generator (WJ-810; Panasonic, Secaucus, NJ) provided on-screen time, date, and stopwatch functions. The images obtained were projected onto a computer monitor (Trinitron 20-in. monitor; Sony, New York, NY). Data were analyzed using MetaMorph 4.5/4.6 (Universal Imaging Corp, Downingtown, Pa).
The rats were divided into three groups: a sham (control) group, an HS group (T60), and an HS group pretreated with α-lipoic acid (100 mg/kg) 10 min before the shock period. This dose of α-lipoic acid has been previously shown to protect against LPS-induced lung injury and associated oxidative stress in rats (22). The experimental groups consisted of five rats each. A 30-min stabilization period was given to allow the animals to recover from surgical manipulation before the start of all experiments, followed by the recording of baseline parameters: MAP, red blood cell centerline velocity, and vessel diameter. During this period, the animals were given FITC-albumin (50 mg/kg) and baseline-integrated optical intensities were obtained intravascularly and extravascularly (two sites, same computed areas, the mean values were used). To produce HS, the MAP was decreased to 40 mmHg by withdrawing blood from the right carotid artery into a syringe containing 100 units of heparin. To obtain this level of HS, it requires approximately 50% to 60% of the animal's blood volume, level IV shock. This period was taken as shock T0. After the shock period, the shed blood plus two times the volume of isotonic sodium chloride solution was reinfused to maintain a MAP at or greater than 90 mmHg for 60 min (T60). Mesenteric postcapillary venules in a transilluminated segment of small intestine were examined to quantitate changes in albumin flux. Parameters were recorded postshock at 10-min intervals for 60 min. The following formula was used for the calculation of light intensity: ΔI = Ii - Io/Ii, where ΔI is the change in light intensity, Ii is the light intensity inside the vessel, and Io is the light intensity outside the vessel. Gray-scale values were measured in the postcapillary venules and in the extravascular space adjacent to the predetermined venule using MetaMorph image analysis system as previously described (5).
Mitochondrial ROS formation
Hemorrhagic shock was produced as described previously. The rats were injected i.v. with the fluorescent indicator dihydrorhodamine 123 (50 mg/kg) and were subjected to intravital microscopy. The use of dihydrorhodamine 123 for in vivo visualization of ROS formation in rats was previously published from our laboratory (4). The conversion of dihydrorhodamine 123 to a fluorescent product rhodamine 123 that fluoresce red is positively correlated to mitochondrial ROS formation (23). The changes in fluorescent intensity that indicate ROS formation were evaluated and recorded using automated image analysis.
Mitochondrial transmembrane potential
The rats were allowed to recover from surgical manipulations for 30 min. Each group consisted of five rats. Hemorrhagic shock was induced as described previously. The mesenteric vasculature was superfused with JC-1 reagent (1:100) to measure changes in mitochondrial transmembrane potential using intravital microscopy. The use of JC-1 for determination of the changes in mitochondrial transmembrane potential of rats in vivo was previously published from our laboratory (5). In healthy cells, the cationic fluorescent indicator JC-1 fluoresces the mitochondria red (Cy3 filter, emission wave length 590 nM) and cytoplasm fluoresces green (FITC filter; emission wave length, 530 nM). The negative charge established by the mitochondrial transmembrane potential allows the lipophilic dye, bearing a delocalized positive charge to enter the mitochondrial matrix where it accumulates. When the critical concentration is exceeded, J aggregates form that fluoresce red. In cells undergoing apoptosis, the mitochondrial membrane potential collapses, and the JC-1 cannot accumulate within the mitochondria. In these cells, JC-1 remains in the cytoplasm in a green fluorescent monomeric form.
Cytochrome c release
The rats were divided into the following groups: sham-control, HS groups (T0 and T60), and HS groups (T0 and T60) treated with α-lipoic acid (i.v.; 100 mg/kg) 10 min before shock. Experimental groups consisted of five rats each. Hemorrhagic shock was produced as described previously. Cytochrome c assay from the cytosolic fraction of the rat mesenteric tissue was performed by enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, Minn). The tissues were weighed and homogenized in a cold preparation buffer. The tissue homogenates were centrifuged (10,000g for 60 min at 4°C), and the supernatant (cytosolic fraction) was collected and subjected to protein assay. The samples were treated with a conjugate reagent, transferred to an antibody-coated microwell plate, and incubated for 60 min at room temperature. The wells were washed using a wash solution. The samples were then treated with a peroxidase substrate reagent and incubated for 15 min at room temperature. After the addition of a stop solution, the samples were read at 450 nm within 30 min. A serial dilution of cytochrome c standard was subjected to the assay along with the samples, and the values were plotted on semilog. The values of samples were determined based on the standard plot.
Caspase 3 activity
Hemorrhagic shock was produced in rats as described previously. Caspase 3 activity was determined using a fluorescent method (Calbiochem, La Jolla, Calif) where active caspase 3 cleaves after aspartate residues in a particular peptide sequence (DEVD). The DEVD substrate was labeled with a fluorescent molecule, 7-amino-4-trifluoromethyl coumarin. The mesenteric microvessels harvested from the animals were homogenized in caspase 3 sample lysis buffer provided in the kit. The homogenates were centrifuged at 10,000g for 20 min at 4°C. The resulting supernatant was used for protein estimation and caspase 3 assays. The tissue lysates were treated with the substrate conjugate, and the resulting fluorescent intensity was measured in a fluorescent plate reader at an excitation of 400 nm and an emission of 505 nm. The data are expressed as a percentage of the sham-control values.
All data are expressed as mean ± SE. Statistical analysis was performed using ANOVA followed by the Bonferroni posttest for multiple comparisons. In vascular hyperpermeability studies, each experimental value was compared with initial baseline value and expressed as percentage change. This method decreases bias between animals because of red blood cell accumulation and changes in room lighting. Student t test was also used to test statistical significance between two groups wherever required. A P < 0.05 was considered to indicate a significant difference.
α-Lipoic acid attenuated vascular hyperpermeability
Hemorrhagic shock induced hyperpermeability as evident from the significant extravasation of FITC-albumin fluorescent intensity into the extravascular space over time compared with the sham-control group (P < 0.05; Fig. 1, A and B). α-Lipoic acid (100 mg/kg) given before the HS period resulted in significant attenuation of hyperpermeability compared with the HS group without α-lipoic acid treatment (P < 0.05; Fig. 1, A and B). Figure 1A is a composite image of a rat's mesenteric postcapillary venule before HS demonstrating minimal extravasation of FITC-albumin into the extravascular space. The second image taken from a rat after 60 min of HS and 60 min of resuscitation (T60) demonstrates extravasation of FITC-albumin into the extravascular space. The third image corresponds to α-lipoic acid treatment before the HS period for 60 min and 60 min of resuscitation (T60). α-Lipoic acid significantly attenuated the extravasation of FITC-albumin into the extravascular space. Figure 1B is the graphic representation of the changes in vascular permeability in sham-control and HS with or without α-lipoic acid pretreatment. α-Lipoic acid significantly attenuated HS-induced hyperpermeability (P < 0.05) when given 10 min before the HS period.
α-Lipoic acid decreased mitochondrial ROS formation
Hemorrhagic shock resulted in a significant increase in ROS formation compared with the sham-control group (P < 0.05; Fig. 2, A and B). α-Lipoic acid (100 mg/kg) treatment 10 min before the HS period resulted in significant attenuation of ROS formation compared with the HS group without α-lipoic acid treatment (P < 0.05; Fig. 2, A and B). In Figure 2A, the first image is a composite image of a rat mesenteric postcapillary venule from preshock period demonstrating minimal ROS formation. The second image taken from a rat after 60 min of HS and 60 min of resuscitation (T60) after the shock period (T60) demonstrates increased ROS formation. The third image in Figure 3 represents attenuation of ROS formation in α-lipoic acid pretreatment followed by HS. Figure 2B is the graphic representation of the changes in ROS formation in sham-control and HS with or without α-lipoic acid pretreatment.
α-Lipoic acid decreased mitochondrial transmembrane depolarization
Under normal conditions, the inner mitochondrial membrane is nearly impermeable to maintain inner transmembrane potential. Opening of the permeability pores results in immediate dissipation of the membrane potential with consequent loss of cytochrome c. We used a novel approach to monitor mitochondrial membrane potential in vivo with JC-1 as described previously (5). In nonapoptotic cells, JC-1 exists as a monomer in the cytosol (green) and also accumulates as J aggregates in the mitochondria, which fluoresce as red. Upon the induction of apoptosis, JC-1 exists only in a monomeric form (green). Figure 3 is an image of a mesenteric postcapillary venule from a sham-control rat demonstrating both green (cytosol) and red (mitochondria) fluorescence in the vascular endothelial cells. After shock (T60), there was a decrease in red (mitochondrial) fluorescence indicating the loss of mitochondrial transmembrane potential. α-Lipoic acid (100 mg/kg) pretreatment prevented the loss of mitochondrial transmembrane potential evident from the increase in red fluorescence compared with the HS group without α-lipoic acid treatment.
α-Lipoic acid decreased mitochondrial cytochrome c release
Cytochrome c release from the mitochondria has been reported to be the key event in apoptosis induced by various stimuli. Mesenteric vascular tissue levels of cytosolic cytochrome c were measured from the sham-control group, HS groups (T0 and at T60), and α-lipoic acid-treated HS groups (T0 and at T60; Fig. 4). The cytochrome c level was elevated at shock T0 (68.8 ± 5.8 ng/mg protein) and T60 (177 ± 11.7 ng/mg protein) compared with the sham-control group (21.6 ± 1.6 ng/mg protein) (P < 0.05). α-Lipoic acid (100 mg/kg)-pretreated HS groups (T0 and T60) showed significantly low levels of cytochrome c compared with the HS groups without α-lipoic acid treatment (T0, 35.1 ± 2.7 ng/mg protein; T60, 39± 4.3 ng/mg protein; P < 0.05; Fig. 4).
α-Lipoic acid attenuated caspase 3 activation
Mitochondrial release of cytochrome c leads to the activation of caspase 3. Activation of caspase 3 was determined from rat mesenteric vasculature. The HS group showed a significant increase in caspase 3 activity compared with the sham-control group (T0, 154% ± 7.3% of control and T60, 206% ± 9.6% of control; P < 0.05; Fig. 5). The HS group (T60) that was treated with α-lipoic acid before shock showed a significantly low caspase 3 activity compared with the HS group without α-lipoic acid pretreatment (T60, 123.13% ± 7.6% of control; P < 0.05; Fig. 5).
This study supports our hypothesis that α-lipoic acid attenuates vascular hyperpermeability after HS by inhibiting mitochondrial ROS formation and activation of intrinsic apoptotic signaling. It has been shown that mitochondrial ROS formation, activation of intrinsic apoptotic signaling, and vascular hyperpermeability occur after HS (4, 5). We have now demonstrated that vascular hyperpermeability, mitochondrial ROS formation, mitochondrial membrane depolarization, mitochondrial release of cytochrome c, and activation of caspase 3 that occur after HS were effectively attenuated by α-lipoic acid. These observations suggest that the α-lipoic acid-mediated attenuation of vascular hyperpermeability is caused by its protective effects on mitochondrial transition pores, therefore preventing cytochrome c release and subsequent caspase 3 activation. This study supports our previous hypothesis that intrinsic apoptotic signaling induces vascular hyperpermeability after HS. Furthermore, the study suggests that selective inhibition of this pathway is important in regulating vascular hyperpermeability.
α-Lipoic acid has been previously shown to prevent apoptotic cell death (11, 12, 24). An increase in antiapoptotic Bcl-2 in rat endothelial cells has been recently reported after α-lipoic acid treatment (25). Recent studies have shown that α-lipoic acid attenuated hepatic I/R injury and helped liver regeneration in rats (14) and protected against warm ischemia (13). However, the effects of α-lipoic acid on vascular endothelial barrier functions and the underlying cellular and physiological mechanisms are not known.
Previous work from our laboratory demonstrated a decrease in mitochondrial transmembrane potential, release of cytochrome c from mitochondria, and activation of caspase 3 in association with vascular hyperpermeability after HS (5). These results suggested that an effective approach to control vascular hyperpermeability after HS may be to protect the mitochondrial transition pores and prevent cytochrome c release and subsequent caspase 3 activation. Thus, the main purpose of the present study was to test the effectiveness of an endogenous antioxidant with antiapoptotic properties to protect mitochondrial transition pores. Our results suggest that α-lipoic acid protected mitochondrial transition pores, which is evident from the prevention of leakage of JC-1 dye from mitochondria into the cytoplasm. The observation that α-lipoic acid prevented HS-induced cytochrome c release further shows the protective effects of α-lipoic acid on mitochondrial transition pores. Various mitochondrial functions, including ion transport, adenosine triphosphate formation, and so on, require an intact mitochondrial transmembrane potential that depends upon the generation of electrochemical proton gradient across the mitochondrial inner membrane. A crucial event that occurs in response to apoptotic signaling is loss of this electrochemical proton gradient followed by collapse of mitochondrial transmembrane potential (26). This collapse occurs because of opening of mitochondrial transition pores in the inner mitochondrial membrane that allow the nonselective diffusion of solutes across the membrane. The release of mitochondrial cytochrome c to the cytoplasm takes place through this regulatory mechanism. The present study shows that α-lipoic acid can protect mitochondrial transition pores and thereby prevent cytochrome c release after HS. The cellular mechanisms by which α-lipoic acid interacted with mitochondrial transition pores to prevent cytochrome c release are not clearly known. Mitochondria are the major intracellular source of oxidizing free radicals. These oxidants generated selectively damage mitochondrial macromolecules and mitochondrial membrane functions. α-Lipoic acid might have given protection to mitochondrial membrane by preventing oxidative stress-induced damage of mitochondrial macromolecules and membrane. In normal cells, cytochrome c is located in the mitochondrial intermembrane/intercristae spaces, where it functions as an electron shuttle in the mitochondrial respiratory chain and interacts with cardiolipin (27). The mitochondrial outer membrane permeabilization that occurs in response to several proapoptotic stimuli can mobilize cytochrome c from cardiolipin leading to the release of cytochrome c to the cytosol. α-Lipoic acid treatment has been shown to restore mitochondrial cardiolipin content and membrane potential in aging rats (28). After the release to the cytosol, cytochrome c mediates the allosteric activation of apoptotic protease activating factor 1, which is required for the proteolytic maturation of caspase 9 and caspase 3 (17, 27). So, the caspase 3 activation observed in our study may be explained as a result of an increased mitochondrial release of cytochrome c that occurred after mitochondrial transition pore opening.
Our results show that α-lipoic acid inhibited HS-induced mitochondrial ROS formation. α-Lipoic acid is known to inhibit oxidative stress and directly bind to free radicals (10). An important mediator of HS-induced mitochondrial transition pore opening could be mitochondrial ROS formation and resulting oxidative stress. Oxidative stress is one of the most important mediators of apoptotic signaling and subsequent cell death (9), and has been implicated in HS (4). The I/R damages in endothelial cells and the selective endothelial cell dysfunction occur through a mechanism that involves oxygen-derived free radicals in rat mesentery (29). Continuous mitochondrial oxidative stress caused by ROS after reperfusion injury is known to regulate mitochondrial release of cytochrome c (30). The mitochondrial transition pore is known to be activated by Ca2+ and ROS (20, 21). Exogenous ROS also induced mitochondrial transition pore opening and cytochrome c release in isolated mitochondria (31). Reactive oxygen species and caspase 3 induced cytochrome c release from mitochondria, which could be prevented by dominant-negative caspase 9, caspase inhibitors, and overexpression of Bcl-2 (31), indicating that the ROS formation is associated with increased mitochondrial transition pore opening, cytochrome c release, and caspase 3 activation (31). It is quite possible that in HS-induced vascular permeability, mitochondrial oxidative stress played an important role in the release of cytochrome c to the cytoplasm. α-Lipoic acid, by its antioxidant activity, might have prevented this effect.
The activation of caspase 3 leads to the cleavage of a variety of cell adhesion proteins (17). The major components of endothelial cell adherens junctions are the cadherin family of proteins, α-, β-, and γ-catenins. A stable cell-cell adherens junction requires the close interaction of the cytoplasmic domain of the cadherins with a group of intracellular proteins, the catenins (32). β-Catenin, a member of the Armadillo repeat protein family, functions as a regulator of cadherin-mediated cell-cell adhesion in endothelial cells, and its absence may lead to fluid leakage (33). Proteolytic cleavage of β-catenin occurs after the activation of procaspase 3, 6, or 8 (16, 17, 34, 35). Cleavage of β-catenin was found to be caspase dependent, and five cleavage products of β-catenin were identified in vivo and after in vitro cleavage by caspase 3 (36). Thus, α-lipoic acid mediated inhibition of caspase 3 activation, and subsequent prevention of microvascular endothelial cell-cell detachment is one of the possible mechanisms by which it protected barrier integrity and prevented vascular permeability in HS. Although our results provide supporting evidence for the protective role of α-lipoic acid against hyperpermeability, further studies may be required to establish the direct involvement of α-lipoic acid in preventing endothelial cell-cell detachment and hyperpermeability.
The main purpose of this study was to prove our hypothesis that the interruption of the intrinsic apoptotic signaling cascade using an endogenously present antioxidant and antiapoptotic agent (α-lipoic acid) would attenuate vascular hyperpermeability after HS. In our study, α-lipoic acid showed protective effects against hyperpermeability when it was given before the shock period and supported our hypothesis. The pretreatment strategy of α-lipoic acid used in this study has limitations in a clinical context. The findings of this study will be clinically relevant when α-lipoic acid is administered during or after HS. Our ongoing and future researches will focus on these aspects.
In conclusion, our findings show that α-lipoic acid attenuates vascular hyperpermeability after HS. The protective effect of α-lipoic acid on vascular barrier functions may be caused by its inhibitory effects on ROS formation and intrinsic apoptotic signaling pathway. By its ability to protect mitochondrial transition pores, α-lipoic acid may prevent cytochrome c release and caspase 3 activation. α-Lipoic acid may be further tested for its clinical efficacy to control vascular hyperpermeability that occurs after HS.
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I/R; vascular permeability; mitochondrial membrane potential; cytochrome c; caspase 3; reactive oxygen species