Red Light Mitigates the Deteriorating Placental Extracellular Matrix in Late Onset of Preeclampsia and Improves the Trophoblast Behavior.

Preeclampsia is a serious pregnancy disorder which in extreme cases may lead to maternal and fetal injury or death. Preexisting conditions which increase oxidative stress, e.g., hypertension and diabetes, increase the mother's risk to develop preeclampsia. Previously, we established that when the extracellular matrix is exposed to oxidative stress, trophoblast function is impaired, and this may lead to improper placentation. We investigated how the oxidative ECM present in preeclampsia alters the behavior of first trimester extravillous trophoblasts. We demonstrate elevated levels of advanced glycation end products (AGE) and lipid oxidation end product 4-hydroxynonenal in preeclamptic ECM (28%, and 32% increase vs control, respectively) accompanied with 35% and 82% more 3-chlorotyrosine and 3-nitrotyrosine vs control, respectively. Furthermore, we hypothesized that 670 nm phototherapy, which has antioxidant properties, reverses the observed trophoblast dysfunction as depicted in the improved migration and reduction in apoptosis. Since NO is critical for placentation, we examined eNOS activity in preeclamptic placentas compared to healthy ones and found no differences; however, 670 nm light treatment triggered enhanced NO availability presumably by using alternative NO sources. Light exposure decreased apoptosis and restored trophoblast migration to levels in trophoblasts cultured on preeclamptic ECM. Moreover, 670 nm irradiation restored expression of Transforming Growth Factor (TGFß) and Placental Growth Factor (PLGF) to levels observed in trophoblasts cultured on healthy placental ECM. We conclude the application of 670 nm light can successfully mitigate the damaged placental microenvironment of late onset preeclampsia as depicted by the restored trophoblast behavior.

Abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in a murine model of systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by oxidative stress, impaired vascular function, and attenuated angiogenesis. The tight-skin (Tsk(-/+)) mouse is a model of SSc that displays many of the cellular features of the clinical disease. We tested the hypotheses that abnormal fibrillin-1 expression and chronic phospholipid oxidation occur in Tsk(-/+) mice and, furthermore, that these factors precipitate a prooxidant state, collagen-related protein expression, apoptosis, and mesenchymal transition in endothelial cells cultured on Tsk(-/+) extracellular matrix. Human umbilical vein endothelial cells were seeded on microfibrils isolated from skin of C57BL/6J (control) and Tsk(-/+) mice in the presence or absence of chronic pretreatment with the apolipoprotein Apo A-I mimetic D-4F (1 mg·kg(-1)·day(-1) ip for 6 to 8 wk). Nitric oxide-to-superoxide anion ratio was assessed 12 h after culture, and cell proliferation, apoptosis, and phenotype were studied 72 h after culture. Tsk(-/+) mice demonstrated abnormal "big fibrillin" expression (405 kDa) by Western blot analysis compared with control. Endothelial cells cultured on microfibrils prepared from Tsk(-/+) mice demonstrated reduced proliferation, a prooxidant state (reduced nitric oxide-to-superoxide anion ratio), increased apoptosis, and collagen-related protein expression associated with mesenchymal transition. Chronic D-4F pretreatment of Tsk(-/+) mice attenuated many of these adverse effects. The findings demonstrate that abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in Tsk(-/+) mice. This mesenchymal transition may contribute to the reduction in angiogenesis that is known to occur in this model of SSc.

Red light stimulates vasodilation through extracellular vesicle trafficking.

Red light (670 nm) promotes ex vivo dilation of blood vessels in a nitric oxide (NO) dependent, but eNOS independent manner by secreting a quasi-stable and transferable vasoactive substance with the characteristics of S-nitrosothiols (RSNO) from the endothelium. In the present work we establish that 670 nm light mediated vasodilation occurs in vivo and is physiologically stable. Light exposure depletes intracellular S-nitroso protein while concomitantly increasing extracellular RNSO, suggesting vesicular pathways are involved. Furthermore, we demonstrate this RSNO vasodilator is embedded in extracellular vesicles (EV). The action of red light on vesicular trafficking appears to increase expression of endosome associated membrane protein CD63 in bovine aortic endothelial cells, enhance endosome localization in the endothelium, and induce exit of RSNO containing EVs from murine facialis arteries. We suggest a mechanism by which the concerted actions of 670 nm light initiate formation of RSNO containing EVs which exit the endothelium and trigger relaxation of smooth muscle cells.

Non-thermal Infrared Light Treatment of Ischemia/Reperfusion Injury and Subsequent Analysis of Macrophage Differentiation.

Tissue damage and necrosis from inflammatory processes are a consequence of ischemia reperfusion injury (IRI). In skeletal muscle, ischemia reduces the aerobic energy capacity of muscle cells, leading to adverse biochemical alterations and inflammation. The goal of this study is to show that exposure to near-infrared light (NIR) during a period of ischemia reduces IRI by decreasing necrosis and inflammation in addition to decreasing proinflammatory M1 and increasing protective M2 macrophages. C57/Bl6 mice underwent unilateral tourniquet-induced hindlimb ischemia for 3 h followed by reperfusion for either 15 or 30 min. Mice were randomly assigned to 3 groups. Group 1 underwent IRI with 30 min reperfusion. Group 2 underwent IRI with a 15 min reperfusion. Each group consisted of 50% no-NIR and 50% NIR-treated mice with exposure of 50 mW/cm2 for 5 min/1 h after tourniquet closure. Group 3 were sham animals anesthetized for 3 h omitting IRI. Laser doppler flow imaging was performed on all mice to confirm ischemia and reperfusion. Flow data were expressed as the ratio of ischemic limb and the contralateral control. The mice were euthanized after reperfusion, and the quadriceps and gastrocnemius were harvested. Immunoprecipitation and western blot of macrophage-markers CD68 (M1) and CD206 (M2) were performed and normalized to CD14 expression. The expression of the inflammatory markers CXCL1 and CXCL5 was significantly reduced by NIR in the IRI group. A significant decrease in CD68 and an increase in CD206 expression was observed in animals receiving IR and NIR. Tissue necrosis was decreased by NIR in the IRI group, as visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The findings demonstrate that exposure to NIR reduced IRI and improved tissue survival. NIR reduced inflammation, decreased proinflammatory M1, and increased protective M2 macrophages. Exposure to NIR reduced inflammation and enhanced regeneration, leading to tissue protection following ischemia.

Intralipid Increases Nitric Oxide Release from Human Endothelial Cells During Oxidative Stress.

BACKGROUND: Intralipid (ILP), a lipid emulsion, protects organs against ischemia/reperfusion (IR) injury. We hypothesized that ILP activates endothelial nitric oxide synthase (eNOS) and increases NO release from endothelial cells (ECs) through a fatty-acid translocase cluster of differentiation (CD36) mediated endocytotic mechanism, acting as a potentially protective paracrine signal during oxidative stress. METHODS: Human umbilical-vein ECs were exposed to 1% ILP for 2 hours followed by oxidative stress with 0.2-mM hydrogen peroxide for 2 hours. Western blots were conducted with anti-CD36, dynamin-2, src-kinase-1, eNOS, and phospho-eNOS; equal protein loading was confirmed with ß-actin. CD36 immunoprecipitation was probed for caveolin-1 to determine if CD36 and caveolin-1 were complexed on the cell membrane. NO was measured by fluorescence of ECs. RESULTS: ILP caused a 227% increase in CD36 expression vs controls. Immunoprecipitation indicated a CD36/caveolin-1 complex on ECs' membrane with exposure to ILP. Dynamin-2 increased 52% and src-kinase-1 340% after ILP treatment vs control cells. eNOS phosphorylation was confirmed by a 63% increase in the phospho-eNOS/eNOS ratio in ILP-treated cells, and NO fluorescence increased 102%. CONCLUSION: ILP enters ECs via endocytosis by a CD36/caveolin-1 cell membrane receptor complex, which in turn is pulled into the cell by dynamin-2 activity. Upregulation of src-kinase-1 and eNOS phosphorylation suggest downstream mediators. Subsequent NO release from ECs serve as a paracrine signal to neighboring cells for protection against IR injury. Student t-test was utilized for single comparisons and analysis of variance with Bonferroni-Dunn post hoc modification for multiple comparisons; P < .05 was considered statistically significant.

Inhibition of myeloperoxidase increases revascularization and improves blood flow in a diabetic mouse model of hindlimb ischaemia.

OBJECTIVE: Diabetes mellitus is a significant risk factor for peripheral artery disease. Diabetes mellitus induces chronic states of oxidative stress and vascular inflammation that increase neutrophil activation and release of myeloperoxidase. The goal of this study is to determine whether inhibiting myeloperoxidase reduces oxidative stress and neutrophil infiltration, increases vascularization, and improves blood flow in a diabetic murine model of hindlimb ischaemia. METHODS: Leptin receptor-deficient (db/db) mice were subjected to hindlimb ischaemia. Ischaemic mice were treated with N-acetyl-lysyltyrosylcysteine-amide (KYC) to inhibit myeloperoxidase. After ligating the femoral artery, effects of treatments were determined with respect to hindlimb blood flow, neutrophil infiltration, oxidative damage, and the capability of hindlimb extracellular matrix to support human endothelial cell proliferation and migration. RESULTS: KYC treatment improved hindlimb blood flow at 7 and 14 days in db/db mice; decreased the formation of advanced glycation end products, 4-hydroxynonenal, and 3-chlorotyrosine; reduced neutrophil infiltration into the hindlimbs; and improved the ability of hindlimb extracellular matrix from db/db mice to support endothelial cell proliferation and migration. CONCLUSION: These results demonstrate that inhibiting myeloperoxidase reduces oxidative stress in ischaemic hindlimbs of db/db mice, which improves blood flow and reduces neutrophil infiltration such that hindlimb extracellular matrix from db/db mice supports endothelial cell proliferation and migration.

Role of heat shock protein 90 and endothelial nitric oxide synthase during early anesthetic and ischemic preconditioning.

BACKGROUND: Nitric oxide is known to be essential for early anesthetic preconditioning (APC) and ischemic preconditioning (IPC) of myocardium. Heat shock protein 90 (Hsp90) regulates endothelial nitric oxide synthase (eNOS) activity. In this study, the authors tested the hypothesis that Hsp90-eNOS interactions modulate APC and IPC. METHODS: Myocardial infarct size was measured in rabbits after coronary occlusion and reperfusion in the absence or presence of preconditioning within 30 min of isoflurane (APC) or 5 min of coronary artery occlusion (IPC), and with or without pretreatment with geldanamycin or radicicol, two chemically distinct Hsp90 inhibitors, or N-nitro-L-arginine methyl ester, a nonspecific nitric oxide synthase NOS inhibitor. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells or mouse cardiomyocytes, in the absence or presence of Hsp90 inhibitors or N-nitro-L-arginine methyl ester. Interactions between Hsp90 and eNOS, and eNOS activation, were assessed with immunoprecipitation, immunoblotting, and confocal microscopy. RESULTS: APC and IPC decreased infarct size (by 50% and 59%, respectively), and this action was abolished by Hsp90 inhibitors. N-nitro-L-arginine methyl ester blocked APC but not IPC. Isoflurane increased nitric oxide production in human coronary artery endothelial cells concomitantly with an increase in Hsp90-eNOS interaction (immunoprecipitation, immunoblotting, and immunohistochemistry). Pretreatment with Hsp90 inhibitors abolished isoflurane-dependent nitric oxide production and decreased Hsp90-eNOS interactions. Isoflurane did not increase nitric oxide production in mouse cardiomyocytes, and eNOS was below the level of detection. CONCLUSION: The results indicate that Hsp90 plays a critical role in mediating APC and IPC through protein-protein interactions, and suggest that endothelial cells are important contributors to nitric oxide-mediated signaling during APC.

Transient metabolic alkalosis during early reperfusion abolishes helium preconditioning against myocardial infarction: restoration of cardioprotection by cyclosporin A in rabbits.

BACKGROUND: Intracellular acidosis during early reperfusion after coronary artery occlusion was recently linked to cardioprotection resulting from myocardial ischemic postconditioning. We tested the hypotheses that transient alkalosis during early reperfusion abolishes helium preconditioning and that the mitochondrial permeability transition pore inhibitor cyclosporin A (CsA) restores the cardioprotective effects of helium during alkalosis in vivo. METHODS: Rabbits (n = 36) instrumented for hemodynamics measurement were subjected to a 30-min left anterior descending coronary artery occlusion and 3-h reperfusion. The rabbits received 0.9% saline (control) or three cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture before left anterior descending coronary artery occlusion in the absence or presence of transient alkalosis (pH = 7.5) produced by administration of IV sodium bicarbonate (10 mEq) 2 min before reperfusion. Other rabbits preconditioned with helium received CsA (5 mg/kg) in the presence of alkalosis or CsA alone. RESULTS: Helium reduced myocardial infarct size (25% +/- 4% of left ventricular area at risk; P < 0.05) compared with control (44% +/- 6%). Alkalosis during early reperfusion did not alter infarct size alone (46% +/- 2%), but this intervention abolished helium-induced cardioprotection (45% +/- 3%). CsA restored reductions in infarct size produced by helium preconditioning in the presence of alkalosis (28% +/- 6%; P < 0.05 versus control) but did not affect myocardial necrosis alone (43% +/- 6%). CONCLUSIONS: The results demonstrate that transient alkalosis during early reperfusion abolishes helium preconditioning in rabbits. CsA restored helium-induced cardioprotection during alkalosis, suggesting that helium preconditioning inhibits mitochondrial permeability transition pore formation by maintaining intracellular acidosis during early reperfusion.

Morphine reduces the threshold of helium preconditioning against myocardial infarction: the role of opioid receptors in rabbits.

OBJECTIVES: Brief, repetitive administration of helium before prolonged coronary artery occlusion and reperfusion protects myocardium against infarction. Opioid receptors mediate the cardioprotective effects of ischemic pre- and postconditioning, but whether these receptors also play a role in helium preconditioning is unknown. The authors tested the hypotheses that opioid receptors mediate helium preconditioning and that morphine (a mu(1)-opioid receptor agonist with delta(1)-opioid agonist properties) lowers the threshold of cardioprotection produced by helium in vivo. DESIGN: A randomized, prospective study. SETTING: A university research laboratory. PARTICIPANTS: Male New Zealand white rabbits. INTERVENTIONS: Rabbits (n = 56) were instrumented for the measurement of systemic hemodynamics and subjected to a 30-minute left anterior descending coronary artery (LAD) occlusion and 3 hours of reperfusion. In separate experimental groups, rabbits (n = 6 or 7 per group) received 0.9% saline (control), 1 or 3 cycles of 70% helium-30% oxygen administered for 5 minutes interspersed with 5 minutes of an air-oxygen mixture, morphine (0.1 mg/kg intravenously), or the nonselective opioid antagonist naloxone (6 mg/kg intravenously) before LAD occlusion. Other groups of rabbits received 3 cycles of helium or 1 cycle of helium plus morphine (0.1 mg/kg) in the absence or presence of naloxone (6 mg/kg) before ischemia and reperfusion. Statistical analysis of data was performed with analysis of variance for repeated measures followed by Bonferroni modification of the Student t test. MEASUREMENTS AND MAIN RESULTS: Myocardial infarct size was determined by using triphenyltetrazolium chloride staining and presented as a percentage of the left ventricular area at risk. Helium reduced myocardial infarct size in an exposure-related manner (36 +/- 6 [p > 0.05] and 25% +/- 4% [p < 0.05 v control] for 1 and 3 cycles of helium, respectively; data are mean +/- standard deviation) compared with control (44% +/- 7%). Morphine and naloxone alone did not affect infarct size (45 +/- 2 and 40% +/- 8%, respectively). The combination of 1 cycle of helium and morphine reduced infarct size (24% +/- 5%, p < 0.05 v control) to an equivalent degree as 3 cycles of helium. Naloxone pretreatment abolished cardioprotection produced by 3 cycles of helium (47% +/- 2%) and the combination of 1 cycle of helium plus morphine (45% +/- 4%). CONCLUSIONS: The results indicate that morphine lowers the threshold of helium preconditioning. Opioid receptors mediate helium preconditioning and its augmentation by morphine in vivo.

Inputs to medullary respiratory neurons from a pontine subregion that controls breathing frequency.

Neurons in a subregion of the medial parabrachial (PB) complex control expiratory duration (TE) and the inspiratory on-switch. To better understanding the underlying mechanisms, this study aimed to determine the types of medullary neurons in the rhythmogenic preBötzinger/Bötzinger Complex (preBötC/BötC) and adjacent areas that receive synaptic inputs from the PB subregion and whether these inputs are excitatory or inhibitory in nature. Highly localized electrical stimuli in the PB subregion combined with multi-electrode recordings from respiratory neurons and phrenic nerve activities were used to generate stimulus-to-spike event histograms to detect correlations in decerebrate, vagotomized dogs during isocapnic hyperoxia. Short-time scale correlations were found in 237/442 or ∼54% of the ventral respiratory column (VRC) neurons. Inhibition of E-neurons was ∼2.5X greater than for I-neurons, while Pre-I and I-neurons were excited. These findings indicate that the control of TE and the inspiratory on-switch by the PB subregion are mediated by a marked inhibition of BötC E-neurons combined with an excitation of I-neurons, especially pre-I neurons.

Simvastatin restores ischemic preconditioning in the presence of hyperglycemia through a nitric oxide-mediated mechanism.

BACKGROUND: A growing body of evidence indicates that statins decrease perioperative cardiovascular risk and that these drugs may be particularly efficacious in diabetes. Diabetes and hyperglycemia abolish the cardioprotective effects of ischemic preconditioning (IPC). The authors tested the hypothesis that simvastatin restores the beneficial effects of IPC during hyperglycemia through a nitric oxide-mediated mechanism. METHODS: Myocardial infarct size was measured in dogs (n = 76) subjected to coronary artery occlusion and reperfusion in the presence or absence of hyperglycemia (300 mg/dl) with or without IPC in separate groups. Additional dogs received simvastatin (20 mg orally daily for 3 days) in the presence or absence of IPC and hyperglycemia. Other dogs were pretreated with N-nitro-l-arginine methyl ester (30 mg intracoronary) with or without IPC, hyperglycemia, and simvastatin. RESULTS: Ischemic preconditioning significantly (P < 0.05) reduced infarct size (n = 7, 7 +/- 2%) as compared with control (n = 7, 29 +/- 3%). Hyperglycemia (n = 7), simvastatin (n = 7), N-nitro-l-arginine methyl ester alone (n = 7), and simvastatin with hyperglycemia (n = 6) did not alter infarct size. Hyperglycemia (n = 7, 24 +/- 2%), but not N-nitro-l-arginine methyl ester (n = 5, 10 +/- 1%), blocked the protective effects of IPC. Simvastatin restored the protective effects of IPC in the presence of hyperglycemia (n = 7, 14 +/- 1%), and this beneficial action was blocked by N-nitro-l-arginine methyl ester (n = 7, 29 +/- 4%). CONCLUSIONS: The results indicate that simvastatin restored the cardioprotective effects of IPC during hyperglycemia by nitric oxide-mediated signaling. The results also suggest that enhanced cardioprotective signaling could be a mechanism for statin-induced decreases in perioperative cardiovascular risk.

The mechanism of helium-induced preconditioning: a direct role for nitric oxide in rabbits.

BACKGROUND: Helium produces preconditioning against myocardial infarction by activating prosurvival signaling, but whether nitric oxide (NO) generated by endothelial NO synthase plays a role in this phenomenon is unknown. We tested the hypothesis that NO mediates helium-induced cardioprotection in vivo. METHODS: Rabbits (n = 62) instrumented for hemodynamic measurement were subjected to a 30-min left anterior descending coronary artery occlusion and 3 h reperfusion, and received 0.9% saline (control) or three cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture before left anterior descending coronary artery occlusion in the absence or presence of pretreatment with the nonselective NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME; 10 mg/kg), the selective inducible NOS inhibitor aminoguanidine hydrochloride (AG; 300 mg/kg), or selective neuronal NOS inhibitor 7-nitroindazole (7-NI; 50 mg/kg). In additional rabbits, the fluorescent probe 4,5-diaminofluroscein diacetate (DAF-2DA) and confocal laser microscopy were used to detect NO production in the absence or presence of helium with or without L-NAME pretreatment. RESULTS: Helium reduced (P < 0.05) infarct size (24% +/- 4% of the left ventricular area at risk; mean +/- sd) compared with control (46% +/- 3%). L-NAME, AG, and 7-NI did not alter myocardial infarct size when administered alone. L-NAME, but not 7-NI or AG, abolished helium-induced cardioprotection. Helium enhanced DAF-2DA fluorescence compared with control (26 +/- 8 vs 15 +/- 5 U, respectively). Pretreatment with L-NAME abolished these helium-induced increases in DAF-2DA fluorescence. CONCLUSIONS: The results indicate that cardioprotection by helium is mediated by NO that is probably generated by endothelial NOS in vivo.

Inhibition of glycogen synthase kinase or the apoptotic protein p53 lowers the threshold of helium cardioprotection in vivo: the role of mitochondrial permeability transition.

BACKGROUND: Prosurvival signaling kinases inhibit glycogen synthase kinase-3beta (GSK-3beta) activity and stimulate apoptotic protein p53 degradation. Helium produces cardioprotection by activating prosurvival kinases, but whether GSK and p53 inhibition mediate this process is unknown. We tested the hypothesis that inhibition of GSK or p53 lowers the threshold of helium cardioprotection via a mitochondrial permeability transition pore (mPTP)-dependent mechanism. METHODS: Rabbits (n = 85) instrumented for hemodynamic measurement and subjected to a 30 min left anterior descending coronary artery (LAD) occlusion and 3 h reperfusion received 0.9% saline (control), or 1, 3, or 5 cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture (fraction of inspired oxygen concentration = 0.30) before LAD occlusion. Other rabbits received the GSK inhibitor SB 216763 (SB21; 0.2 or 0.6 mg/kg), the p53 inhibitor pifithrin-alpha (PIF; 1.5 or 3.0 mg/kg), or SB21 (0.2 mg/kg) or PIF (1.5 mg/kg) plus helium (1 cycle) before LAD occlusion in the presence or absence of the mPTP opener atractyloside (5 mg/kg). RESULTS: Helium reduced (P < 0.05) myocardial infarct size (35 +/- 6 [n = 7], 25 +/- 4 [n = 7], and 20 +/- 3% [n = 6] of area at risk, 1, 3, and 5 cycles, respectively) compared with control (44 +/- 6% [n = 7]). SB21 (0.6 [n = 7] but not 0.2 mg/kg [n = 6]) and PIF (3.0 [n = 6] but not 1.5 mg/kg [n = 7]) also reduced necrosis. SB21 (0.2 mg/kg) or 1.5 mg/kg PIF (1.5 mg/kg) plus helium (1 cycle; n = 6 per group) decreased infarct size to an equivalent degree as three cycles of helium alone, and this cardioprotection was blocked by atractyloside (n = 7 per group). CONCLUSIONS: Inhibition of GSK or p53 lowers the threshold of helium-induced preconditioning via a mPTP-dependent mechanism in vivo.

Reactive oxygen species and mitochondrial adenosine triphosphate-regulated potassium channels mediate helium-induced preconditioning against myocardial infarction in vivo.

OBJECTIVES: Helium produces preconditioning by activating prosurvival kinases, but the roles of reactive oxygen species (ROS) or mitochondrial adenosine triphosphate-regulated potassium (K(ATP)) channels in this process are unknown. The authors tested the hypothesis that ROS and mitochondrial K(ATP) channels mediate helium-induced preconditioning in vivo. DESIGN: A randomized, prospective study. SETTING: A university research laboratory. PARTICIPANTS: Male New Zealand white rabbits. INTERVENTIONS: Rabbits (n = 64) were instrumented for the measurement of systemic hemodynamics and subjected to a 30-minute left anterior descending coronary artery (LAD) occlusion and 3 hours of reperfusion. In separate experimental groups, rabbits (n = 7 or 8 per group) were randomly assigned to receive 0.9% saline (control) or 3 cycles of 70% helium-30% oxygen administered for 5 minutes interspersed with 5 minutes of an air-oxygen mixture before LAD occlusion with or without the ROS scavengers N-acetylcysteine (NAC; 150 mg/kg) or N-2 mercaptoproprionyl glycine (2-MPG; 75 mg/kg), or the mitochondrial K(ATP) antagonist 5-hydroxydecanoate (5-HD; 5 mg/kg). Statistical analysis of data was performed with analysis of variance for repeated measures followed by Bonferroni's modification of a Student t test. MEASUREMENTS AND MAIN RESULTS: The myocardial infarct size was determined by using triphenyltetrazolium chloride staining and presented as a percentage of the left ventricular area at risk. Helium significantly (p < 0.05) reduced infarct size (23 +/- 4% of the area at risk; mean +/- standard deviation) compared with control (46 +/- 3%). NAC, 2-MPG, and 5-HD did not affect irreversible ischemic injury when administered alone (49 +/- 5%, 45 +/- 6%, and 45 +/- 3%), but these drugs blocked reductions in infarct size produced by helium (45 +/- 4%, 45 +/- 2%, and 44 +/- 3%). CONCLUSIONS: The results suggest that ROS and mitochondrial K(ATP) channels mediate helium-induced preconditioning in vivo.

Noble gases without anesthetic properties protect myocardium against infarction by activating prosurvival signaling kinases and inhibiting mitochondrial permeability transition in vivo.

BACKGROUND: The anesthetic noble gas, xenon, produces cardioprotection. We hypothesized that other noble gases without anesthetic properties [helium (He), neon (Ne), argon (Ar)] also produce cardioprotection, and further hypothesized that this beneficial effect is mediated by activation of prosurvival signaling kinases [including phosphatidylinositol-3-kinase, extracellular signal-regulated kinase, and 70-kDa ribosomal protein s6 kinase] and inhibition of mitochondrial permeability transition pore (mPTP) opening in vivo. METHODS: Rabbits (n = 98) instrumented for hemodynamic measurement and subjected to a 30-min left anterior descending coronary artery (LAD) occlusion and 3 h reperfusion received 0.9% saline (control), three cycles of 70% He-, Ne-, or Ar-30% O2 administered for 5 min interspersed with 5 min of 70% N2-30% O2 before LAD occlusion, or three cycles of brief (5 min) ischemia interspersed with 5 min reperfusion before prolonged LAD occlusion and reperfusion (ischemic preconditioning). Additional groups of rabbits received selective inhibitors of phosphatidylinositol-3-kinase (wortmannin; 0.6 mg/kg), extracellular signal-regulated kinase (PD 098059; 2 mg/kg), or 70-kDa ribosomal protein s6 kinase (rapamycin; 0.25 mg/kg) or mPTP opener atractyloside (5 mg/kg) in the absence or presence of He pretreatment. RESULTS: He, Ne, Ar, and ischemic preconditioning significantly (P < 0.05) reduced myocardial infarct size [23% +/- 4%, 20% +/- 3%, 22% +/- 2%, 17% +/- 3% of the left ventricular area at risk (mean +/- sd); triphenyltetrazolium chloride staining] versus control (45% +/- 5%). Wortmannin, PD 098059, rapamycin, and atractyloside alone did not affect infarct size, but these drugs abolished He-induced cardioprotection. CONCLUSIONS: The results indicate that noble gases without anesthetic properties produce cardioprotection by activating prosurvival signaling kinases and inhibiting mPTP opening in rabbits.

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice.

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides. IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 ± 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 µg/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 µg/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

Inhibition of apoptotic protein p53 lowers the threshold of isoflurane-induced cardioprotection during early reperfusion in rabbits.

INTRODUCTION: Exposure to isoflurane before and during early reperfusion protects against myocardial infarction by activating phosphatidylinositol-3-kinase (PI3K)-mediated signaling. The apoptotic protein, p53, is regulated by PI3K, and inhibition of p53 protects against ischemic injury. We tested the hypothesis that p53 inhibition lowers the threshold of isoflurane-induced postconditioning in vivo. METHODS: Rabbits (n = 73) instrumented for hemodynamic measurement and subjected to a 30-min left anterior descending coronary artery occlusion and 3-h reperfusion received 0.9% saline (control), isoflurane (0.5 or 1.0 minimum alveolar concentration [MAC]) administered for 3 min before and 2 min after reperfusion, the p53 inhibitor pifithrin-alpha (1.5 or 3.0 mg/kg), or 0.5 MAC isoflurane plus 1.5 mg/kg pifithrin-alpha. Other rabbits received 3.0 mg/kg pifithrin-alpha or 0.5 MAC isoflurane plus 1.5 mg/kg pifithrin-alpha after pretreatment with the selective PI3K inhibitor wortmannin (0.6 mg/kg) or the mitochondrial permeability transition pore opener atractyloside (5 mg/kg). RESULTS: Isoflurane (1.0 but not 0.5 MAC), pifithrin-alpha (3.0 but not 1.5 mg/kg), and the combination of 0.5 MAC isoflurane plus 1.5 mg/kg pifithrin-alpha significantly (P < 0.05) reduced infarct size (21% +/- 4%, 43% +/- 7%, 22% +/- 4%, 45% +/- 4%, and 28% +/- 3% [mean +/- sd], respectively, of left ventricular area at risk; triphenyltetrazolium chloride staining) when compared with control (45% +/- 2%). Atractyloside, but not wortmannin, abolished 3.0 mg/kg pifithrin-alpha-induced cardioprotection, whereas atractyloside and wortmannin blocked reductions in infarct size produced by 0.5 MAC isoflurane plus 1.5 mg/kg pifithrin-alpha. CONCLUSION: The results indicate that inhibition of the apoptotic protein p53 lowers the threshold of isoflurane-induced cardioprotection during early reperfusion in vivo.

Gender-specificity of delayed preconditioning by isoflurane in rabbits: potential role of endothelial nitric oxide synthase.

INTRODUCTION: Female gender confers cardioprotection against ischemia-reperfusion injury, in part because estrogen enhances nitric oxide production by endothelial nitric oxide synthase (eNOS). Whether ischemic preconditioning occurs in females remains controversial. Delayed myocardial preconditioning by isoflurane is mediated by eNOS in male rabbits, but whether females are similarly protected by isoflurane is unknown. We tested the hypothesis that gender-specific reductions in myocardial infarct size occur in female rabbits, but that this inherent cardioprotection abrogates further beneficial effects of isoflurane-induced delayed preconditioning. METHODS: Rabbits (n = 115) underwent a 30 min coronary artery occlusion and 3 h reperfusion with or without a 2 h administration of 1.0 minimum alveolar concentration isoflurane one day before experimentation. Rabbits received saline or a nonselective, selective inducible, or selective neuronal NOS inhibitor [N-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg), aminoguanidine (AG, 300 mg/kg), or 7-nitroindazole (7-NI, 50 mg/kg), respectively]. RESULTS: Isoflurane reduced infarct size in males (mean+/- sd, 26 +/- 5% of the left ventricular area at risk) versus saline (45 +/- 2%). L-NAME, but not AG or 7-NI, abolished isoflurane-induced protection in males (41 +/- 9, 24 +/- 4 and 22 +/- 2%, respectively). Infarct size was reduced, and eNOS protein expression was greater, in female versus male rabbits. Infarct size was unchanged in female rabbits with, versus without, isoflurane pretreatment (27 +/- 9 and 27 +/- 10%, respectively). L-NAME, but not AG or 7-NI, increased infarct size with or without isoflurane pretreatment in females. CONCLUSIONS: Female gender-induced reductions in infarct size are mediated by eNOS, but remote isoflurane exposure (1.0 MAC) before ischemia and reperfusion does not produce additional cardioprotection in vivo.

Inhibition of glycogen synthase kinase enhances isoflurane-induced protection against myocardial infarction during early reperfusion in vivo.

Inhibition of glycogen synthase kinase (GSK)-beta protects against ischemia-reperfusion injury. Brief exposure to isoflurane before and during early reperfusion after coronary artery occlusion also protects against infarction. Whether GSK-beta mediates this action is unknown. We tested the hypothesis that GSK inhibition enhances isoflurane-induced postconditioning. Rabbits (n = 88; 6 to 7 per group) subjected to a 30-min coronary occlusion followed by 3 h reperfusion received saline, isoflurane (0.5 or 1.0 minimum alveolar concentration [MAC]) administered for 3 min before and 2 min after reperfusion, the selective GSK inhibitor SB216763 (SB21; 0.2 or 0.6 mg/kg), or 0.5 MAC isoflurane plus 0.2 mg/kg SB21. Other groups of rabbits pretreated with phosphatidylinositol-3 kinase (PI3K) inhibitor wortmannin (0.6 mg/kg), 70-kDa ribosomal protein s6 kinase (p70s6K) inhibitor rapamycin (0.25 mg/kg), or mitochondrial permeability transition pore (mPTP) opener atractyloside (5 mg/kg) received 0.6 mg/kg SB21 or 0.5 MAC isoflurane plus 0.2 mg/kg SB21. Additional groups received the mPTP inhibitor, cyclosporin A (5 mg/kg), plus 0.2 mg/kg SB21 with or without atractyloside pretreatment. Isoflurane (1.0 but not 0.5 MAC) and SB21 (0.6 but not 0.2 mg/kg) reduced (P < 0.05) infarct size (21% +/- 5%, 44% +/- 7%, 23% +/- 4%, and 46% +/- 2%, respectively, of left ventricular area at risk, mean+/- sd; triphenyltetrazolium staining) as compared with control (42% +/- 6%). Isoflurane (0.5 MAC) plus 0.2 mg/kg SB21 and cyclosporin A plus 0.2 mg/kg SB21 produced similar degrees of protection (24% +/- 4% and 27% +/- 6%, respectively). Atractyloside but not wortmannin or rapamycin abolished protection produced by 0.6 mg/kg SB21 and 0.5 MAC isoflurane plus 0.2 mg/kg SB21. Thus, GSK inhibition enhances isoflurane-induced protection against infarction during early reperfusion via a mPTP-dependent mechanism.

The influence of B-cell lymphoma 2 protein, an antiapoptotic regulator of mitochondrial permeability transition, on isoflurane-induced and ischemic postconditioning in rabbits.

Brief exposure to isoflurane or repetitive, transient ischemia during early reperfusion after prolonged coronary artery occlusion protects against myocardial infarction by inhibiting the mitochondrial permeability transition pore (mPTP). Inhibition of mPTP during delayed ischemic preconditioning occurred concomitant with enhanced expression of the antiapoptotic protein B cell lymphoma-2 (Bcl-2). We tested the hypothesis that Bcl-2 mediates myocardial protection by isoflurane or brief ischemic episodes during reperfusion in rabbits (n = 91) subjected to a 30-min left anterior descending coronary artery occlusion followed by 3 h reperfusion. Rabbits received 0.9% saline, isoflurane (0.5 or 1.0 minimum alveolar concentration, MAC) administered for 3 min before and 2 min after reperfusion, 3 cycles of postconditioning ischemia (10 or 20 s each) during early reperfusion, 0.5 MAC isoflurane plus 3 cycles of postconditioning ischemia (10 s), or the direct mPTP inhibitor cyclosporin A (CsA, 10 mg/kg) in the presence or absence of the selective Bcl-2 inhibitor HA14-1 (2 mg/kg, i.p.). Isoflurane (1.0, but not 0.5, MAC) and postconditioning ischemia (20 s but not 10 s) significantly (P < 0.05) reduced infarct size (mean +/- sd, 21% +/- 4%, 43% +/- 7%, 19% +/- 7%, and 39% +/- 11%, respectively, of left ventricular area at risk) as compared with control (44% +/- 4%). Isoflurane (0.5 MAC) plus 10 s postconditioning ischemia and CsA alone also exerted protection. HA14-1 alone did not affect infarct size nor block protection produced by CsA but abolished reductions in infarct size caused by 1.0 MAC isoflurane, 20 s postconditioning ischemia, and 0.5 MAC isoflurane plus 10 s postconditioning ischemia. The results suggest that Bcl-2 mediates isoflurane-induced and ischemic postconditioning by indirectly modulating mPTP activity in vivo.