Dorothee Weihrauch

Ischemia-Induced Coronary Collateral Growth Is Dependent on Vascular Endothelial Growth Factor and Nitric Oxide

Background—We hypothesized that ischemia-induced expression of vascular endothelial growth factor (VEGF) and the production of NO stimulate coronary collateral growth. Methods and Results—To test this hypothesis, we measured coronary collateral blood flow and VEGF expression in myocardial interstitial fluid in a canine model of repetitive myocardial ischemia under control conditions and during antagonism of NO synthase. Collateralization was induced by multiple (1/h; 8/d), brief (2 minutes) occlusions of the left anterior descending coronary artery for 21 days. In controls, collateral blood flow (microspheres) progressively increased to 89±9 mL · min−1 · 100 g−1 on day 21, which was equivalent to perfusion in the normal zone. Reactive hyperemic responses (a measure of the severity of ischemia) decreased as collateral blood flow increased. In NG-nitro-l-arginine methyl ester (L-NAME)– and L-NAME+nifedipine–treated dogs, to block the production of NO and control hypertension, respectively, collateral blood flow did not increase and reactive hyperemia was robust throughout the occlusion protocol (P <0.01 versus control). VEGF expression (Western analyses of VEGF164 in myocardial interstitial fluid) in controls peaked at day 3 of the repetitive occlusions but waned thereafter. In sham-operated dogs (instrumentation but no occlusions), expression of VEGF was low during the entire protocol. In contrast, VEGF expression was elevated throughout the 21 days of repetitive occlusions after L-NAME. Reverse transcriptase–polymerase chain reaction analyses revealed that the predominant splice variant expressed was VEGF164. Conclusions—NO is an important regulator of coronary collateral growth, and the expression of VEGF is induced by ischemia. Furthermore, the induction of coronary collateralization by VEGF appears to require the production of NO.

Mechanism of Preconditioning by Isoflurane in Rabbits: A Direct Role for Reactive Oxygen Species

Background Reactive oxygen species (ROS) contribute to myocardial protection during ischemic preconditioning, but the role of the ROS in protection against ischemic injury produced by volatile anesthetics has only recently been explored. We tested the hypothesis that ROS mediate isoflurane-induced preconditioning in vivo. Methods Pentobarbital-anesthetized rabbits were instrumented for measurement of hemodynamics and were subjected to a 30 min coronary artery occlusion followed by 3 h reperfusion. Rabbits were randomly assigned to receive vehicle (0.9% saline), or the ROS scavengers N-acetylcysteine (NAC; 150 mg/kg) or N-2-mercaptopropionyl glycine (2-MPG; 1 mg · kg−1· min−1), in the presence or absence of 1.0 minimum alveolar concentration (MAC) isoflurane. Isoflurane was administered for 30 min and then discontinued 15 min before coronary artery occlusion. A fluorescent probe for superoxide anion production (dihydroethidium, 2 mg) was administered in the absence of the volatile anesthetic or 5 min before exposure to isoflurane in 2 additional groups (n = 8). Myocardial infarct size and superoxide anion production were assessed using triphenyltetrazolium staining and confocal fluorescence microscopy, respectively. Results Isoflurane (P < 0.05) decreased infarct size to 24 ± 4% (mean ± SEM; n = 10) of the left ventricular area at risk compared with control experiments (43 ± 3%; n = 8). NAC (43 ± 3%; n = 7) and 2-MPG (42 ± 5%; n = 8) abolished this beneficial effect, but had no effect on myocardial infarct size (47 ± 3%; n = 8 and 46 ± 3; n = 7, respectively) when administered alone. Isoflurane increased superoxide anion production as compared with control experiments (28 ± 12 vs. −6 ± 9 fluorescence units;P < 0.05). Conclusions The results indicate that ROS produced following administration of isoflurane contribute to protection against myocardial infarction in vivo.

L-4F, an Apolipoprotein A-1 Mimetic, Dramatically Improves Vasodilation in Hypercholesterolemia and Sickle Cell Disease

Background—Hypercholesterolemia and sickle cell disease (SCD) impair endothelium-dependent vasodilation by dissimilar mechanisms. Hypercholesterolemia impairs vasodilation by a low-density lipoprotein (LDL)–dependent mechanism. SCD has been characterized as a chronic state of inflammation in which xanthine oxidase (XO) from ischemic tissues increases vascular superoxide anion (O2·−) generation. Recent reports indicate that apolipoprotein (apo) A-1 mimetics inhibit atherosclerosis in LDL receptor–null (Ldlr−/−) mice fed Western diets. Here we hypothesize that L-4F, an apoA-1 mimetic, preserves vasodilation in hypercholesterolemia and SCD by decreasing mechanisms that increase O2·− generation. Methods and Results—Arterioles were isolated from hypercholesterolemic Ldlr−/− mice and from SCD mice that were treated with either saline or L-4F (1 mg/kg per day). Vasodilation in response to acetylcholine was determined by videomicroscopy. Effects of L-4F on LDL-induced increases in endothelium-dependent O2·− generation were determined on arterial segments via the hydroethidine assay and on stimulated endothelial cell cultures via superoxide dismutase–inhibitable ferricytochrome c reduction. Effects of L-4F on XO bound to pulmonary arterioles and content in livers of SCD mice were determined by immunofluorescence. Hypercholesterolemia impaired vasodilation in Ldlr−/− mice, which L-4F dramatically improved. L-4F inhibited LDL-induced increases in O2·− in arterial segments and in stimulated cultures. SCD impaired vasodilation, increased XO bound to pulmonary endothelium, and decreased liver XO content. L-4F dramatically improved vasodilation, decreased XO bound to pulmonary endothelium, and increased liver XO content compared with levels in untreated SCD mice. Conclusions—These data show that L-4F protects endothelium-dependent vasodilation in hypercholesterolemia and SCD. Our findings suggest that L-4F restores vascular endothelial function in diverse models of disease and may be applicable to treating a variety of vascular diseases.

Angiostatin Inhibits Coronary Angiogenesis During Impaired Production of Nitric Oxide

Background—The in vivo mechanism by which inhibition of NO synthase impairs ischemia-induced coronary vascular growth is unknown. We hypothesized that production of the growth inhibitor angiostatin increases during decreased NO production, blunting angiogenesis and collateral growth. Methods and Results—Measurements were made in myocardial tissue or interstitial fluid (MIF) from dogs undergoing repetitive coronary occlusions under control conditions or during antagonism of NO synthase (NG-nitro-l-arginine methyl ester [L-NAME]) for 7, 14, or 21 days. A sham group was instrumented identically but received no occlusions. In controls, capillary density in the ischemic zone increased initially but returned to baseline at the later times. In the L-NAME group, capillary density was lower at 7 days compared with that of controls. MIF from control dogs induced in vitro endothelial tube formation and cell proliferation, significantly greater than that from the L-NAME group. MIF from shams did not stimulate tube formation. In controls or shams, tube formation or cell proliferation did not change after administration of antiangiostatin, but this antibody restored the responses to control levels in the L-NAME group. Angiostatin expression in MIF was increased in the L-NAME group compared with controls and shams. The activities of tissue matrix metalloproteinases (MMPs) MMP-2 and MMP-9, which generate angiostatin, were increased in the L-NAME group. Conclusions—Inhibition of NO synthase increased expression of angiostatin and activities of MMP-2 and MMP-9. Our findings indicate that angiostatin inhibits coronary angiogenesis during compromised NO production and may underscore the impairment of coronary angiogenesis during endothelial dysfunction.

Mitochondrial adenosine triphosphate-regulated potassium channel opening acts as a trigger for isoflurane-induced preconditioning by generating reactive oxygen species

Background Whether the opening of mitochondrial adenosine triphosphate–regulated potassium (KATP) channels is a trigger or an end effector of anesthetic-induced preconditioning is unknown. We tested the hypothesis that the opening of mitochondrial KATP channels triggers isoflurane-induced preconditioning by generating reactive oxygen species (ROS) in vivo. Methods Pentobarbital-anesthetized rabbits were subjected to a 30-min coronary artery occlusion followed by 3 h reperfusion. Rabbits were randomly assigned to receive a vehicle (0.9% saline) or the selective mitochondrial KATP channel blocker 5-hydroxydecanoate (5-HD) alone 10 min before or immediately after a 30-min exposure to 1.0 minimum alveolar concentration (MAC) isoflurane. In another series of experiments, the fluorescent probe dihydroethidium was used to assess superoxide anion production during administration of 5-HD or the ROS scavengers N-acetylcysteine or N-2-mercaptopropionyl glycine (2-MPG) in the presence or absence of 1.0 MAC isoflurane. Myocardial infarct size and superoxide anion production were measured using triphenyltetrazolium staining and confocal fluorescence microscopy, respectively. Results Isoflurane (P < 0.05) decreased infarct size to 19 ± 3% (mean ± SEM) of the left ventricular area at risk as compared to the control (38 ± 4%). 5-HD administered before but not after isoflurane abolished this beneficial effect (37 ± 4% as compared to 24 ± 3%). 5-HD alone had no effect on infarct size (42 ± 3%). Isoflurane increased fluorescence intensity. Pretreatment with N-acetylcysteine, 2-MPG, or 5-HD before isoflurane abolished increases in fluorescence, but administration of 5-HD after isoflurane only partially attenuated increases in fluorescence produced by the volatile anesthetic agent. Conclusions The results indicate that mitochondrial KATP channel opening acts as a trigger for isoflurane-induced preconditioning by generating ROS in vivo.

Inhibition of mitochondrial permeability transition enhances isoflurane-induced cardioprotection during early reperfusion: the role of mitochondrial KATP channels.

Inhibition of the mitochondrial permeability transition pore (mPTP) mediates the protective effects of brief, repetitive ischemic episodes during early reperfusion after prolonged coronary artery occlusion. Brief exposure to isoflurane immediately before and during early reperfusion also produces cardioprotection, but whether mPTP is involved in this beneficial effect is unknown. We tested the hypothesis that mPTP mediates isoflurane-induced postconditioning and also examined the role of mitochondrial KATP (mKATP) channels in this process. Rabbits (n = 102) subjected to a 30-min coronary occlusion followed by 3 h reperfusion received 0.9% saline (control), isoflurane (0.5 or 1.0 MAC) administered for 3 min before and 2 min after reperfusion, or the mPTP inhibitor cyclosporin A (CsA, 5 or 10 mg/kg) in the presence or absence of the mPTP opener atractyloside (5 mg/kg) or the selective mKATP channel antagonist 5-hydroxydecanoate (5-HD; 10 mg/kg). Other rabbits received 0.5 MAC isoflurane plus 5 mg/kg CsA in the presence and absence of atractyloside or 5-HD. Isoflurane (1.0 but not 0.5 MAC) and CsA (10 but not 5 mg/kg) reduced (P < 0.05) infarct size (21% ± 4%, 44% ± 6%, 24% ± 3%, and 43% ± 6%, respectively, mean ± sd of left ventricular area at risk; triphenyltetrazolium staining) as compared with control (42% ± 7%). Isoflurane (0.5 MAC) plus CsA (5 mg/kg) was also protective (27% ± 4%). Neither atractyloside nor 5-HD alone affected infarct size, but these drugs abolished protection by 1.0 MAC isoflurane, 10 mg/kg CsA, and 0.5 MAC isoflurane plus 5 mg/kg CsA. The results indicate that mPTP inhibition enhances, whereas opening abolishes, isoflurane-induced postconditioning. This isoflurane-induced inhibition of mitochondrial permeability transition is dependent on activation of mitochondrial KATP channels 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.

Protein kinase C translocation and Src protein tyrosine kinase activation mediate isoflurane-induced preconditioning in vivo : potential downstream targets of mitochondrial adenosine triphosphate-sensitive potassium channels and reactive oxygen species

BackgroundThe authors tested the hypotheses that protein kinase C (PKC)–specific isoform translocation and Src protein tyrosine kinase (PTK) activation play important roles in isoflurane-induced preconditioning in vivo. MethodsRats (n = 125) instrumented for measurement of hemodynamics underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion and received 0.9% saline (control); PKC inhibitors chelerythrine (5 mg/kg), rottlerin (0.3 mg/kg), or PKC-&egr;V1–2 peptide (1 mg/kg); PTK inhibitors lavendustin A (1 mg/kg) or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1; 1 mg/kg); mitochondrial adenosine triphosphate–sensitive potassium channel antagonist 5-hydroxydecanote (10 mg/kg); or reactive oxygen species scavenger N-acetylcysteine (150 mg/kg) in the absence and presence of a 30-min exposure to isoflurane (1.0 minimum alveolar concentration) in separate groups. Isoflurane was discontinued 15 min before coronary occlusion (memory period). Infarct size was determined using triphenyltetrazolium staining. Immunohistochemistry and confocal microscopic imaging were performed to examine PKC translocation in separate groups of rats. ResultsIsoflurane significantly (P < 0.05) reduced infarct size (40 ± 3% [n = 13]) as compared with control experiments (58 ± 2% [n = 12]). Chelerythrine, rottlerin, PKC-&egr;V1–2 peptide, lavendustin A, PP1, 5-hydroxydecanote, and N-acetylcysteine abolished the anti-ischemic actions of isoflurane (58 ± 2% [n = 8], 50 ± 3% [n = 9], 53 ± 2% [n = 9], 59 ± 3% [n = 6], 57 ± 3% [n = 7], 60 ± 3% [n = 7], and 53 ± 3% [n = 6], respectively). Isoflurane stimulated translocation of the &dgr; and &egr; isoforms of PKC to sarcolemmal and mitochondrial membranes, respectively. ConclusionsProtein kinase C-&dgr;, PKC-&egr;, and Src PTK mediate isoflurane-induced preconditioning in the intact rat heart. Opening of mitochondrial adenosine triphosphate–sensitive potassium channels and generation of reactive oxygen species are upstream events of PKC activation in this signal transduction process.

Morphine Enhances Isoflurane-Induced Postconditioning Against Myocardial Infarction: The Role of Phosphatidylinositol-3-Kinase and Opioid Receptors in Rabbits

Isoflurane reduces myocardial infarct size during early reperfusion by activating phosphatidylinositol-3-kinase (PI3K) signaling. We tested the hypothesis that this cardioprotection against reperfusion injury is enhanced by morphine and that a decrease in apoptosis plays a role in preservation of myocardial viability. Rabbits (n = 108) instrumented for hemodynamic measurement and subjected to a 30-min coronary occlusion followed by 3 h reperfusion received 0.9% saline, the selective PI3K inhibitor wortmannin (0.6 mg/kg), or the nonselective opioid antagonist naloxone (6 mg/kg) before coronary occlusion in the presence or absence of isoflurane (0.5 or 1.0 MAC), morphine (0.05 or 0.1 mg/kg), or their combination administered for 3 min before and 2 min after reperfusion. Infarct size was determined using triphenyltetrazolium staining and apoptosis assessed using cytochrome c translocation and Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) staining of left ventricular myocardium in situ. Isoflurane (1.0 but not 0.5 MAC) and morphine (0.1 but not 0.05 mg/kg) reduced (P < 0.05) infarct size (mean ± sd 21% ± 4%, 44% ± 6%, 19% ± 4%, and 41% ± 6% of left ventricular area at risk, respectively) as compared with control (41% ± 4%). The combination of 0.5 MAC isoflurane and 0.05 mg/kg morphine also decreased infarct size (18% ± 9%). Wortmannin and naloxone alone did not affect infarct size but blocked the protection produced by isoflurane, morphine, and their combination. Isoflurane and morphine reduced cytochrome c translocation and TUNEL staining. The results indicate that morphine enhances isoflurane-induced postconditioning by activating PI3K and opioid receptors in vivo. A reduction in apoptotic cell death contributes to preservation of myocardial integrity during postconditioning by isoflurane.

Role of endothelial nitric oxide synthase as a trigger and mediator of isoflurane-induced delayed preconditioning in rabbit myocardium.

Background:Isoflurane produces delayed preconditioning in vivo. The authors tested the hypothesis that endothelial, inducible, or neuronal nitric oxide synthase (NOS) is a trigger or mediator of this protective effect. Methods:In the absence or presence of exposure to isoflurane (1.0 minimum alveolar concentration) 24 h before experimentation, pentobarbital-anesthetized rabbits (n = 128) instrumented for hemodynamic measurement received 0.9% saline (control), the nonselective NOS inhibitor N-nitro-l-arginine methyl ester (10 mg/kg), one of two of the selective inducible NOS antagonists aminoguanidine (300 mg/kg) or 1400W (0.5 mg/kg), or the selective neuronal NOS inhibitor 7-nitroindazole (50 mg/kg) administered before exposure to isoflurane (trigger; day 1) or left anterior descending coronary artery occlusion (mediator; day 2). All rabbits underwent 30 min of coronary occlusion followed by 3 h of reperfusion. Tissue samples for reverse-transcription polymerase chain reaction and immunohistochemistry were also obtained in the presence or absence of N-nitro-l-arginine methyl ester with or without isoflurane pretreatment. Results:Isoflurane significantly (P < 0.05) reduced infarct size (23 ± 5% [mean ± SD] of the left ventricular area at risk; triphenyltetrazolium chloride staining) as compared with control (42 ± 7%). N-nitro-l-arginine methyl ester administered before isoflurane or coronary occlusion abolished protection (49 ± 7 and 43 ± 10%, respectively). Aminoguanidine, 1400W, and 7-nitroindazole did not alter infarct size or affect isoflurane-induced delayed preconditioning. Isoflurane increased endothelial but not inducible NOS messenger RNA transcription and protein translation immediately and 24 h after administration of the volatile agent. Pretreatment with N-nitro-l-arginine methyl ester attenuated isoflurane-induced increases in endothelial NOS expression. Conclusions:The results suggest that endothelial NOS but not inducible or neuronal NOS is a trigger and mediator of delayed preconditioning by isoflurane in vivo.