eNOS phosphorylation: a pivotal molecular switch in vasodilation and cardioprotection?
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Endothelium, Vascular↗
Biomedical subjects
Lei Xi
Publications and source records attributed to Lei Xi.
Hypercholesterolemia enhances tolerance to lethal systemic hypoxia in middle-aged mice: possible role of VEGF downregulation in brain.
Hypercholesterolemia (HCL) is commonly associated with impaired vascular relaxation response and augmented vasoconstriction. Interestingly, it was shown that animals with HCL were less vulnerable to seizures and several clinical studies also revealed a better outcome after stroke in the patients with HCL. To this context, the present study was designed to test the hypothesis that HCL would enhance the animals' resistance to severe systemic hypoxia and in turn prolong their survival time under such noxious condition. Four groups of middle-aged (mean age: 51.1 +/- 2.8 weeks) male C57BL/6J wild-type mice (C57BL-WT) and low-density lipoprotein receptor knockout mice (LDLR-KO) were included in the study: two groups were exposed to severe normobaric hypoxia (5% F(I)O(2)) and other two groups were used for brain tissue sample collection and Western blot analysis. The survival time under the hypoxic condition was recorded for each animal. Individual blood samples were collected immedtately after the cessation of spontaneous breathing for measuring plasma total cholesterol (TCL) and triglycerides. The results show that the hypoxia survival time was longer in LDLR-KO than C57BL-WT (i.e. 3.7 +/- 0.5 versus 2.3 +/- 0.2 min; P < 0.05). A positive correlation was found between TCL and the survival time (r (2) = 0.43; P < 0.05). Furthermore, a significant downregulation of vascular endothelial growth factor (VEGF) was observed in the brain tissue of LDLR-KO, as compared with C57BL-WT (n, = 3/group; P < 0.05), whereas expression of heme oxygenase 1 was similar in these two groups. We conclude that HCL enhances resistance to lethal systemic hypoxia (i.e. 61% increase in survival time) in middle-aged mice. This paradoxical protective effect of HCL was associated with a concomitant downregulation of cerebral VEGF expression, which could potentially blunt the hypoxia-triggered and VEGF-mediated pathophysiological events leading to death.
Animals↗
Rapamycin confers preconditioning-like protection against ischemia-reperfusion injury in isolated mouse heart and cardiomyocytes.
Rapamycin (sirolimus) is an antibiotic that inhibits protein synthesis through mammalian target of rapamycin (mTOR) signaling and is used as an immunosuppressant in the treatment of organ rejection in transplant recipients. Recently, the antigrowth properties of rapamycin have been utilized for cardiovascular benefit as stents impregnated with rapamycin effectively reduce coronary restenosis. We report here a novel role of this drug in protection against ischemia/reperfusion (I/R) injury. Adult male ICR mice were treated with rapamycin (0.25 mg/kg, IP) or volume-matched DMSO (solvent for rapamycin). The hearts were subjected to 20 min of global ischemia and 30 min of reperfusion in Langendorff mode. The blocker of mitochondrial KATP channel, 5-hydroxydecanoate (5-HD, 100 microM) was given 10 min before ischemia. Infarct size in the DMSO treated group was 28.2 +/- 1.3% and was reduced to 10.1 +/- 2.8% in the rapamycin-treated mice (64% decrease, P < 0.001). 5-HD blocked the protective effect (infarct area 32.2 +/- 1.8%, P < 0.001 vs. rapamycin). The infarct limiting effect of rapamycin was not associated with improved recovery of ventricular function. We further examined the effect of rapamycin in protection against necrosis and apoptosis in adult cardiomyocytes subjected to simulated ischemia and reoxygenation. Myocytes treated with rapamycin in doses from 25-100 nM demonstrated significantly lower trypan blue-positive necrotic cells and TUNEL-positive apoptotic nuclei, supporting the protective role of drug in the intact heart. These data suggest that rapamycin induces potent preconditioning-like effect against myocardial infarction through opening of mitochondrial KATP channels. We propose that rapamycin may be a novel therapeutic strategy to limit infarction, apoptosis, and remodeling following I/R injury in the heart.
Animals↗
Genetic deletion of fas receptors or Fas ligands does not reduce infarct size after acute global ischemia-reperfusion in isolated mouse heart.
Apoptosis has been shown in cardiac cells under divergent physiological and pathological conditions. However, there has been an ongoing debate upon the relative contribution of cardiomyocyte apoptosis to the myocardial infarct size after ischemia-reperfusion (I-R) injury. We tested the hypothesis that blocking the death receptor pathway of apoptosis through genetic deletion of Fas receptors or Fas ligands would reduce myocardial infarct size caused by acute I-R injury. The hearts isolated from Fas receptor or Fas ligand knockout (KO) mice as well as the C57BL/6J wild-type control mice (N = 6-8 per group) were subjected to 20 min of global ischemia and 30 min of reperfusion in Langendorff mode. Our results show that the infarct size, determined with triphenyltetrazolium chloride staining, was not significantly different between the three groups (i.e., 30.2 +/- 3.9% for wild-type controls, 30.0 +/- 2.1% for Fas ligand KOs, and 23.8 +/- 3.6% for Fas receptor KOs; mean +/- SEM, p > 0.05). Postischemic leakage of lactate dehydrogenase, another marker of necrotic cellular injury, also was not significantly different between these groups (p > 0.05). In addition, postischemic ventricular contractile function as well as coronary flow were similar for all the experimental groups (p > 0.05). In conclusion, contrary to our original hypothesis, the present study in the gene KO mice suggests that the Fas ligand- and Fas receptor-mediated death receptor pathway of apoptosis is not the primary determinant of myocardial infarct size and ventricular dysfunction caused by acute global I-R injury in the isolated perfused mouse heart.
Animals↗
[Dynamic changes of morphological parameters of tobacco root in field].
The study with rhizotron showed that the total length of tobacco's second order lateral root had a much more increase than that of first order one, and the root length developed rapidly during 26 to approximately 40 d and 56 to approximately 70 d after transplanting for the second and first order lateral root, respectively. Before tip pruning, the branch root density of tobacco was decreased in order of 10 to approximately 20 > 0 to approximately 10 > 20 to approximately 30 > 30 to approximately 40 cm soil layer. The branch density of main root was the highest in 7 to approximately 21 cm soil layer. The deeper the soil layer, the bigger the ratio of dry weight and root length, but the distribution was reverse at 90 d after transplanting. The first order lateral root length density distributed in "S" curve in 0 to approximately 10 cm soil layer and in double climax curve in 10 to approximately 20, 20 to approximately 30 and 30 to approximately 40 cm soil layers, while the second order lateral root length density became bigger with the course of growth period, which was in "S" curve in 0 to approximately 10 cm soil layer and in single climax curve in other soil layers.
Plant Roots↗
Pharmacological preconditioning with sildenafil: Basic mechanisms and clinical implications.
The phosphodiesterase type-5 (PDE5) inhibitor, sildenafil, is the first drug developed for treatment of erectile dysfunction in patients. Experimental data in animals show that sildenafil has a preconditioning-like cardioprotective effect against ischemia/reperfusion injury in the intact heart. Mechanistic studies suggest that sildenafil exerts cardioprotection through NO generated from eNOS/iNOS, activation of protein kinase C/ERK signaling and opening of mitochondrial ATP-sensitive potassium channels. Additional studies show that the drug attenuates cell death resulting from necrosis and apoptosis, and increases the Bcl2/Bax ratio through NO signaling in adult cardiomyocytes. Emerging new data also suggest that sildenafil may be used clinically for treatment of pulmonary arterial hypertension and endothelial dysfunction. Future demonstration of the cardioprotective effect in patients with the relatively safe and effective FDA-approved PDE5 inhibitors such as sildenafil could have an enormous impact on bringing the long-studied phenomenon of ischemic and pharmacologic preconditioning to the clinical forefront.
3',5'-Cyclic-GMP Phosphodiesterases↗
Phosphodiesterase-5 inhibitor sildenafil preconditions adult cardiac myocytes against necrosis and apoptosis. Essential role of nitric oxide signaling.
We investigated the effect of sildenafil in protection against necrosis or apoptosis in cardiomyocytes. Adult mouse ventricular myocytes were treated with sildenafil (1 or 10 microM) for 1 h before 40 min of simulated ischemia (SI). Necrosis was determined by trypan blue exclusion and lactate dehydrogenase release following SI alone or plus 1 or 18 h of reoxygenation (RO). Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and mitochondrial membrane potential measured using a fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Sildenafil reduced necrosis as indicated by decrease in trypan blue-positive myocytes and leakage of lactate dehydrogenase compared with untreated cells after either SI or SI-RO. The number of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive myocytes or loss of JC-1 fluorescence following SI and 18 h of RO was attenuated in the sildenafil-treated group with concomitant inhibition of caspase 3 activity. An early increase in Bcl-2 to Bax ratio with sildenafil treatment was also observed in myocytes after SI-RO. The increase of Bcl-2 expression by sildenafil was inhibited by nitric-oxide synthase (NOS) inhibitor, L-nitro-amino-methyl-ester. The drug also enhanced mRNA and protein content of inducible NOS (iNOS) and endothelial NOS (eNOS) in the myocytes. Sildenafil-induced protection against necrosis and apoptosis was absent in the myocytes derived from iNOS knock-out mice and was attenuated in eNOS knock-out myocytes. The up-regulation of Bcl-2 expression by sildenafil was also absent in iNOS-deficient myocytes. Reverse transcription-PCR, Western blots, and immunohistochemical assay confirmed the expression of phosphodiesterase-5 in mouse cardiomyocytes. These data provide strong evidence for a direct protective effect of sildenafil against necrosis and apoptosis through NO signaling pathway. The results may have possible therapeutic potential in preventing myocyte cell death following ischemia/reperfusion.
3',5'-Cyclic-GMP Phosphodiesterases↗
Silencing heat shock factor 1 by small interfering RNA abrogates heat shock-induced cardioprotection against ischemia-reperfusion injury in mice.
Induction of heat shock factor 1 (HSF1) is known to associate with cellular response to divergent pathophysiological stresses including whole body hyperthermia (WBH) and ischemia-reperfusion. However, a direct cause-effect relationship between HSF1 activation and cytoprotection induced by myocardial preconditioning has not been conclusively established, mainly due to the limitations of available experiment tools. In the present studies, we used a novel approach to block HSF1 with small interfering RNA (siRNA) technique in vivo. Male adult ICR mice were treated intraperitoneally with amine (vehicle) or siRNA specific to HSF1 (siRNA-HSF1). Three days later, WBH preconditioning protocol (rectal temperature 42 degrees C for 15 min) was applied to these mice under light anesthesia. WBH preconditioning resulted in 2.7-fold and 3.4-fold increase in cardiac HSF1 mRNA and protein expression respectively 2 hours after WBH, which was inhibited in the siRNA-treated mice. The silencing effect of siRNA on HSF1 was associated with complete loss of the infarct- limiting protection by WBH preconditioning after 48 hours. Pretreatment with siRNA-HSF1 had no effect on infarct size in the sham control animals as compared with the amine-treated group. DNA micro-array analysis revealed that siRNA-HSF1 caused a general inhibition on multiple members of HSP family, except Hsp32, Hsp47 and Hsp60. In addition, the silencing effect of siRNA on HSF1 and HSPs gene expression was transient and its inhibitory effect disappeared by 10 days after treatment. siRNA-HSF1 also impaired the thermotolerance of the heat shocked mice as indicated by higher mortality following WBH. For the first time, we have applied siRNA technique in the field of myocardial preconditioning to demonstrate HSF1 activation as an essential step in WBH preconditioning against cardiac ischemia-reperfusion injury.
Animals↗
Cobalt chloride induces delayed cardiac preconditioning in mice through selective activation of HIF-1alpha and AP-1 and iNOS signaling.
Acute systemic hypoxia induces delayed cardioprotection against ischemia (I)-reperfusion (R) injury via inducible nitric oxide synthase (iNOS)-dependent mechanism. Because CoCl2 is known to elicit hypoxia-like responses, we hypothesized that this chemical would mimic the delayed preconditioning effect in the heart. Adult male mice were pretreated with CoCl2 or saline. The hearts were isolated 24 h later and subjected to 20 min of global I and 30 min of R in Langendorff mode. Myocardial infarct size (% of risk area; mean +/- SE, n=6-8/group) was reduced in mice pretreated with 30 mg/kg CoCl2 (16.1 +/- 3.1% vs. 27.6 +/- 3.3% with saline control; P <0.05) without compromising postischemic cardiac function. Higher doses of CoCl2 failed to induce similar protection. Electrophoretic mobility gel shift assay demonstrated significant enhancement in DNA binding activity of hypoxia-inducible factor 1alpha (HIF-1alpha) and activator protein 1 (AP-1) in nuclear extracts from CoCl2-treated hearts. Activation of HIF-1alpha and AP-1 was evident at 30 min and sustained for the next 4 h after CoCl2 injection. In contrast, CoCl2-induced protection was independent of NF-kappaB activation because no DNA binding or p65 translocation was observed in nuclear extracts. Also, CoCl2-induced cardioprotection was preserved in p50 subunit NF-kappaB-knockout (KO) mice (11.1 +/- 3.0% vs. 25.1 +/- 5.0% in saline-treated p50-KO mice; P <0.05). The infarct-limiting effect of CoCl2 was absent in iNOS-KO mice (20.9 +/- 3.0%). We conclude that in vivo administration of CoCl2 preconditions the heart against I/R injury. The delayed protective effect of CoCl2 is achieved through a distinctive signaling mechanism involving HIF-1alpha, AP-1, and iNOS but independent of NF-kappaB activation.
Animals↗
Opening of Ca2+-activated K+ channels triggers early and delayed preconditioning against I/R injury independent of NOS in mice.
Opening of Ca2+-activated K+ (KCa) channels has been shown to confer early cardioprotection. It is unknown whether the opening of these channels also induces delayed cardioprotection. In addition, we determined the involvement of nitric oxide synthases (NOSs), which have been implicated in cardioprotection induced by opening of mitochondrial ATP-sensitive K+ (KATP) channels. Adult male ICR mice were pretreated with the KCa-channel opener NS-1619 either 10 min or 24 h before 30 min of global ischemia and 60 min of reperfusion (I/R) in Langendorff mode. Infusion of NS-1619 (10 microM) for 10 min before I/R led to smaller infarct sizes as compared with the vehicle (DMSO)-treated group (P <0.05). This infarct-limiting effect of NS-1619 was associated with improvement in ventricular functional recovery after I/R. The NS-1619-induced protection was abolished by coadministration with the KCa-channel blocker paxilline (1 microM). Similarly, pretreatment with NS-1619 (1 mg/kg ip) induced delayed protection 24 h later (P <0.05). Interestingly, the NS-1619-induced late protection was not blocked by the NOS inhibitor Nomega-nitro-L-arginine methyl ester (15 mg/kg ip). Unlike diazoxide (the opener of mitochondrial KATP channels), NS-1619 did not increase the expression of inducible or endothelial NOS. Western blot analysis demonstrated the existence of alpha- and beta-subunits of KCa channels in mouse heart tissue. We conclude that opening of KCa channels leads to both early and delayed preconditioning effects through a mechanism that is independent of nitric oxide.
Animals↗
Cardioprotection with phosphodiesterase-5 inhibition--a novel preconditioning strategy.
Phosphodiesterase type-5 (PDE-5) inhibitors including sildenafil, vardenafil and tadalafil are a new class of vasoactive drugs that have been developed for treatment of erectile dysfunction in patients. A growing number of studies in recent years suggest that sildenafil may be used clinically for treatment of pulmonary hypertension and endothelial dysfunction. In addition, recent studies primarily from our laboratory suggested that sildenafil has preconditioning-like powerful cardioprotective effect in the animal models of ischemia-reperfusion injury. Sildenafil has been found to exert cardioprotection through nitric oxide generated from endothelial and/or inducible nitric oxide synthases and opening of mitochondrial ATP-sensitive potassium channels. Future demonstration of the cardioprotective effect in patients with the relatively safe and effective FDA-approved PDE-5 inhibitors, such as sildenafil, could have an enormous impact on bringing the long-studied phenomena of ischemic and pharmacologic preconditioning to the clinical forefront.
3',5'-Cyclic-GMP Phosphodiesterases↗
Sildenafil-induced cardioprotection in rabbits.
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Animals↗
Sildenafil induces delayed preconditioning through inducible nitric oxide synthase-dependent pathway in mouse heart.
Sildenafil citrate (Viagra) is the most widely used drug for treating erectile dysfunction in men. We recently demonstrated that it induces potent protective effects against ischemia-reperfusion (I-R) injury in rabbit hearts through the opening of mitochondrial ATP-dependent K+ channels. In the present study, we investigated the role of the NO-dependent signaling pathway in delayed cardioprotection by sildenafil. Adult male ICR mice were treated with saline or sildenafil (0.7 mg/kg IP) 24 hours before global I-R in the Langendorff mode. Infarct size was reduced from 27.6+/-3.3% in saline-treated control mice to 6.9+/-1.2% in sildenafil-treated mice (mean+/-SEM, P<0.05) without compromising cardiac function. Reverse transcription-polymerase chain reaction revealed a transient increase in endothelial and inducible NO synthase (eNOS and iNOS, respectively) mRNA in sildenafil-treated mice, peaking at 45 minutes (eNOS) and 2 hours (iNOS) after sildenafil injection. The magnitude of mRNA increase was more pronounced for iNOS than for eNOS. In addition, a significant increase in both iNOS and eNOS protein was detected 24 hours after sildenafil treatment. A selective inhibitor of iNOS, 1400W (10 mg/kg IP given 30 minutes before I-R), abolished sildenafil-induced protection (23.7+/-2.8%, P<0.05 versus sildenafil). These data suggest that the induction of NO synthase isoforms is an essential component of the signaling mechanism for sildenafil-induced delayed preconditioning. However, iNOS appears to be the primary isoform that mediates the robust cardioprotection.
Animals↗
Exercise does not protect the female heart: an unconvincing conclusion?
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Animals↗
Evidence that NOS2 acts as a trigger and mediator of late preconditioning induced by acute systemic hypoxia.
Chronic systemic hypoxia (SH) enhances myocardial ischemic tolerance in mammals. We studied the delayed cardioprotection caused by acute SH and associated signaling mechanism. Conscious adult male mice were exposed to one or two cycles of hypoxia (H; 10% O(2)) or normoxia (21% O(2)) for various durations (30 min, 2 h, 4 h) followed by 24 h of reoxygenation. Hearts were isolated 24 h later and subjected to ischemia-reperfusion in a Langendorff model. Infarct size was reduced in mice pretreated with one (H4h) or two cycles (H4hx2) of 4 h SH compared with normoxia mice (P < 0.05), which was abolished by an inducible nitric oxide synthase (NOS2) inhibitor (S-methylisothiourea, 3 mg/kg) given before SH or ischemia. H4hx2 also failed to reduce infarct size in NOS2 knockout mice. Cyclooxygenase-2 (COX-2) inhibitor (NS-398, 10 mg/kg) did not block the protection given either before H4hx2 or ischemia. A two- to three fold increase in myocardial NOS2 expression was observed in H4h, H2hx2, and H4hx2 (P < 0.05), whereas endothelial NOS (NOS3) or COX-2 remained unchanged. We conclude that acute SH induces delayed cardioprotection, which is triggered and mediated by NOS2, but not by NOS3 or COX-2.
Animals↗