Breathing nitric oxide plus hydrogen gas reduces ischemia-reperfusion injury and nitrotyrosine production in murine heart.

Inhaled nitric oxide (NO) has been reported to decrease the infarct size in cardiac ischemia-reperfusion (I/R) injury. However, reactive nitrogen species (RNS) produced by NO cause myocardial dysfunction and injury. Because H2 is reported to eliminate peroxynitrite, it was expected to reduce the adverse effects of NO. In mice, left anterior descending coronary artery ligation for 60 min followed by reperfusion was performed with inhaled NO [80 parts per million (ppm)], H2 (2%), or NO + H2, starting 5 min before reperfusion for 35 min. After 24 h, left ventricular function, infarct size, and area at risk (AAR) were assessed. Oxidative stress associated with reactive oxygen species (ROS) was evaluated by staining for 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal, that associated with RNS by staining for nitrotyrosine, and neutrophil infiltration by staining for granulocyte receptor-1. The infarct size/AAR decreased with breathing NO or H2 alone. NO inhalation plus H2 reduced the infarct size/AAR, with significant interaction between the two, reducing ROS and neutrophil infiltration, and improved the cardiac function to normal levels. Although nitrotyrosine staining was prominent after NO inhalation alone, it was eliminated after breathing a mixture of H2 with NO. Preconditioning with NO significantly reduced the infarct size/AAR, but not preconditioning with H2. In conclusion, breathing NO + H2 during I/R reduced the infarct size and maintained cardiac function, and reduced the generation of myocardial nitrotyrosine associated with NO inhalation. Administration of NO + H2 gases for inhalation may be useful for planned coronary interventions or for the treatment of I/R injury.

Synchronization between cardiac and respiratory rhythms in healthy subjects and patients.

Synchronization between cardiac and respiratory rhythms may be important for oxygen transport to tissues. The aim of this study was to investigate the synchronization between cardiac and respiratory rhythms. We evaluated the rhythms in 12 healthy males and 24 patients. The incidence rates of heart beats were obtained in each time interval relative to the initiation time point of inspiration. A simple index of timing variability of heart beats was defined. When the variability is large, the link between cardiac and respiratory rhythms was considered to be strong. The variability value of patients with disorder in the autonomic nervous system was larger than that of healthy subjects (p < 0.05). The variability of patients on controlled ventilation was lower than that of healthy subjects (p < 0.01), whereas the value on cardiac pacemaker did not differ from healthy subjects. In conclusion, the synchronization between cardiac and respiratory rhythms was confirmed, and it is suggested that the synchronization is enhanced when feed-back signals from respiratory movement to respiratory center were decreased.

Successful rechallenge with erlotinib in a patient with EGFR-mutant lung adenocarcinoma who developed gefitinib-related interstitial lung disease.

Small-molecule tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) pathways are used clinically for patients with non-small cell lung cancer (NSCLC). It is well established that somatic mutations in the kinase domain of the EGFR (Lynch et al. in N Engl J Med 350:2129-2139, 2004; Paez et al. in Science 304:1497-1500, 2004) are strongly associated with the tumor response and clinical outcomes in patients with NSCLC receiving EGFR-TKIs (Mitsudomi and Yatabe in Cancer Sci 98:1817-1824, 2007). Although the most common adverse events are skin rash and diarrhea, the most serious adverse effect reported is drug-related interstitial lung disease (ILD) (Inoue et al. in Lancet 361:137-139, 2003; Ando et al. in J Clin Oncol 24:2549-2556, 2006). The precise mechanism underlying the development of drug-related ILD remains unknown. Here, we describe a case of EGFR-mutant NSCLC who was rechallenged with the small-molecule EGFR antagonist erlotinib after developing gefitinib-related ILD.

Effect of gefitinib on warfarin antithrombotic activity.

BACKGROUND: Despite the literature indicating adverse interactions between warfarin and cytotoxic agents, whether such an interaction occurs when warfarin and gefitinib are used concomitantly is unknown. We analyzed the prevalence of the concomitant use of warfarin and gefitinib, and the incidence of prothrombin time-international normalized ratio (PT-INR) alterations or adverse interactions in concomitant users of warfarin and gefitinib. METHODS: We conducted a retrospective study of patients with non-small cell lung cancer treated at the Kitasato University Hospital who received concomitant warfarin and gefitinib between September 2002 and January 2007. Medical information, including the indication for warfarin use, warfarin dosing and dosing changes, and exposure to gefitinib were collected from computerized databases and medical records. RESULTS: Twelve (4.1%) of 296 patients treated with gefitinib received warfarin. PT-INR elevation occurred in 6 patients (50.0%). Two (16.7%) of the 12 patients had liver metastases. Liver dysfunction was associated with PT-INR elevation (P = 0.0100). CONCLUSION: As there is a possibility of PT-INR abnormalities occurring during the concomitant use of gefitinib and warfarin, clinicians should be aware of this interaction. Because of the potentially severe consequences of this interaction, close monitoring of PT-INR and warfarin dose adjustment are recommended for patients receiving warfarin and gefitinib, especially during the first 2 weeks in the beginning of warfarin therapy.

Infarct size assessment in mice.

Genetically modified mice are used extensively in models of ischemia reperfusion (I/R) and nonreperfused myocardial infarction (MI) to gain insights into pathways involved in these pathologies. Echocardiography is an ideal noninvasive tool to serially monitor the cardiac murine phenotype. The present review details the surgical aspects of I/R and MI models and the measurement of MI size by pathology techniques and the input of echocardiographic techniques including the extent of wall motion abnormality and of perfusion defects using myocardial contrast echocardiography in the assessment of murine area at risk and MI size.

Nitric oxide synthase 2 and pressure-overload-induced left ventricular remodelling in mice.

Nitric oxide synthase 2 (NOS2) has been reported to increase in hypertrophied cardiomyocytes; however, whether NOS2 plays a role in the development of hypertrophy is unknown. To investigate the relationship of NOS2 with left ventricular (LV) remodelling and hypertrophy following prolonged pressure overload, we studied 18 male wild-type (WT) and 20 male NOS2-deficient (NOS2-/-) mice before and 7, 14 and 28 days after transverse aortic constriction (TAC) using echocardiography. A subgroup of eight WT and eight NOS2-/- mice were studied 42 days after TAC. Haemodynamic measurements were obtained before killing. Left ventricular size and function were similar for both genotypes at baseline. After TAC for 28 days, both groups developed LV hypertrophy, with echo-derived LV mass increasing from 78 +/- 2 to 147 +/- 10 mg in WT and from 86 +/- 3 to 142 +/- 10 mg in NOS2-/- mice. Twenty-eight days after TAC, LV weight and cardiomyocyte width were also similar in both genotypes. Fractional shortening (FS) decreased on day 7 from 57 +/- 1 to 48 +/- 2% in WT and from 59 +/- 1 to 49 +/- 2% in NOS2-/- mice. Although this decrease in FS was transient in WT mice, it persisted in NOS2-/- mice. Invasively measured parameters of systolic and diastolic function, however, were similar in the two genotypes both 28 and 42 days after TAC. A load-independent index of contractility, Emax, was similar in both strains 42 days after TAC. In conclusion, NOS2 does not appear to have a critical role in the development of LV hypertrophy after chronic pressure overload.

Inhaled nitric oxide decreases infarction size and improves left ventricular function in a murine model of myocardial ischemia-reperfusion injury.

To learn whether nitric oxide (NO) inhalation can decrease myocardial ischemia-reperfusion (I/R) injury, we studied a murine model of myocardial infarction (MI). Anesthetized mice underwent left anterior descending coronary artery ligation for 30, 60, or 120 min followed by reperfusion. Mice breathed NO beginning 20 min before reperfusion and continuing thereafter for 24 h. MI size and area at risk were measured, and left ventricular (LV) function was evaluated using echocardiography and invasive hemodynamic measurements. Inhalation of 40 or 80 ppm, but not 20 ppm, NO decreased the ratio of MI size to area at risk. NO inhalation improved LV systolic function, as assessed by echocardiography 24 h after reperfusion, and systolic and diastolic function, as evaluated by hemodynamic measurements 72 h after reperfusion. Myocardial neutrophil infiltration was reduced in mice breathing NO, and neutrophil depletion prevented inhaled NO from reducing myocardial I/R injury. NO inhalation increased arterial nitrite levels but did not change myocardial cGMP levels. Breathing 40 or 80 ppm NO markedly and significantly decreased MI size and improved LV function after ischemia and reperfusion in mice. NO inhalation may represent a novel method to salvage myocardium at risk of I/R injury.

Inhaled nitric oxide does not reduce systemic vascular resistance in mice.

Inhaled nitric oxide (NO) is a highly selective pulmonary vasodilator. It was recently reported that inhaled NO causes peripheral vasodilatation after treatment with a NO synthase (NOS) inhibitor. These findings suggested the possibility that inhibition of endogenous NOS uncovered the systemic vasodilating effect of NO or NO adducts absorbed via the lungs during NO inhalation. To learn whether inhaled NO reduces systemic vascular resistance in the absence of endothelial NOS, we studied the systemic vascular effects of NO breathing in wild-type mice treated without and with the NOS inhibitor N(omega)-nitro-l-arginine methyl ester and in NOS3-deficient (NOS3(-/-)) mice. During general anesthesia, the cardiac output, left ventricular function, and systemic vascular resistance were not altered by NO breathing at 80 parts/million in both genotypes. Breathing NO in air did not alter blood pressure and heart rate, as measured by tail-cuff and telemetric methods, in either awake wild-type mice (whether or not they were treated with N(omega)-nitro-l-arginine methyl ester), or in awake NOS3(-/-) mice. Our findings suggest that absorption of NO or adducts during NO breathing is insufficient to cause systemic vasodilation in mice, even when endogenous endothelial NO production is congenitally absent.

Quantitative assessment of regional myocardial function in mice by tissue Doppler imaging: comparison with hemodynamics and sonomicrometry.

BACKGROUND: Tissue Doppler imaging (TDI) is a novel echocardiographic method to quantify regional myocardial function. The objective of this study was to assess whether myocardial velocities and strain rate (SR) could be obtained by TDI in mice and whether these indices accurately quantified alterations in left ventricular (LV) systolic function. METHODS AND RESULTS: TDI was performed in 10 healthy mice to measure endocardial (v(endo)) and epicardial systolic velocities and SR. In further experiments, TDI indices were compared with dP/dt(max) and with sonomicrometer-derived regional velocities, at rest and after administration of dobutamine or esmolol. TDI indices were also studied serially in 8 mice before and 4 and 7 hours after endotoxin challenge. Myocardial velocities and SR were obtained in all mice with low measurement variability. TDI indices increased with administration of dobutamine (v(endo) from 2.2+/-0.3 to 3.8+/-0.2 cm/s [P<0.01]; SR from 12+/-2 to 20+/-2 s(-1) [P<0.05]) and decreased with administration of esmolol (v(endo) 1.4+/-0.2 cm/s [P<0.05]; SR 6+/-1 s(-1) [P<0.01]). Both indices correlated strongly with dP/dt(max) (r2=0.79 for SR and r2= 0.69 for v(endo); both P<0.0001). SR and shortening fraction were predictors of dP/dt(max) even after adjustment for the confounding effect of the other variables. V(endo) correlated closely with sonomicrometer-measured velocity (r2=0.71, P<0.0005). After endotoxin challenge, decreases in both v(endo) and SR were detected before decreases in shortening fraction became manifest. CONCLUSIONS: Myocardial velocities and SR can be measured noninvasively in mice with the use of TDI. Both indices are sensitive markers for quantifying LV global and regional function in mice.

Relationship of systolic dysfunction to area at risk and infarction size after ischemia-reperfusion in mice.

Whereas the extent of wall-motion abnormality (WMA) correlates well with the area at risk (AAR) and infarction size in murine models of nonreperfused myocardial infarction, this relationship is less clear in the setting of ischemia-reperfusion injury. Echocardiography was performed in mice at baseline, after left anterior descending coronary artery ligation (30 minutes) followed by reperfusion (24 hours), and after religation of the left anterior descending coronary artery. The extent of WMA before and after religation was compared with the initial infarction size measured by triphenyltetrazolium chloride and the AAR measured by fluorescent microspheres, respectively. Echocardiography showed left ventricular dilation and dysfunction after ischemia and reperfusion. WMA after religation correlated well with AAR (r2 = 0.70, P <.0001). The correlation between WMA and infarction size was incomplete (r2 = 0.59, P <.0002) in part because of underestimation of nontransmural infarcts. Echocardiography can reliably assess AAR after ischemia-reperfusion in mice; however, it does not allow for precise quantification of the small areas of necrosis that often occur in this setting.

Pressure overload-induced LV hypertrophy and dysfunction in mice are exacerbated by congenital NOS3 deficiency.

To investigate the role of endothelial nitric oxide synthase (NOS3) in left ventricular (LV) remodeling induced by chronic pressure overload, the impact of transverse aortic constriction (TAC) on LV structure and function was compared in wild-type (WT) and NOS3-deficient (NOS3(-/-)) mice. Before TAC, LV wall thickness, mass, and fractional shortening were similar in the two mouse strains. Twenty-eight days after TAC, both WT and NOS3(-/-) mice had increased LV wall thickness and mass as well as decreased fractional shortening. Although the pressure gradient across the TAC was similar in both strains of mice 28 days after TAC, LV mass and posterior wall thickness were greater in NOS3(-/-) than in WT mice, whereas fractional shortening and the maximum rate of developed LV pressure were less. Diastolic function, as measured by the time constant of isovolumic relaxation and the maximum rate of LV pressure decay, was impaired to a greater extent in NOS3(-/-) than in WT mice. The degree of myocyte hypertrophy and LV fibrosis was greater in NOS3(-/-) than in WT mice at 28 days after TAC. Mortality was greater in NOS3(-/-) than in WT mice 28 days after TAC. Long-term administration of hydralazine normalized the blood pressure and prevented the LV dilation in NOS3(-/-) mice but did not prevent the LV hypertrophy, dysfunction, and fibrosis associated with NOS3 deficiency after TAC. These results suggest that the absence of NOS3 augments LV dysfunction and remodeling in a murine model of chronic pressure overload.

A selective inducible NOS dimerization inhibitor prevents systemic, cardiac, and pulmonary hemodynamic dysfunction in endotoxemic mice.

Increased nitric oxide (NO) production by inducible NO synthase (NOS2), an obligate homodimer, is implicated in the cardiovascular sequelae of sepsis. We tested the ability of a highly selective NOS2 dimerization inhibitor (BBS-2) to prevent endotoxin-induced systemic hypotension, myocardial dysfunction, and impaired hypoxic pulmonary vasoconstriction (HPV) in mice. Mice were challenged with Escherichia coli endotoxin before treatment with BBS-2 or vehicle. Systemic blood pressure was measured before and 4 and 7 h after endotoxin challenge, and echocardiographic parameters of myocardial function were measured before and 7 h after endotoxin challenge. The pulmonary vasoconstrictor response to left mainstem bronchus occlusion, which is a measure of HPV, was studied 22 h after endotoxin challenge. BBS-2 treatment alone did not alter baseline hemodynamics. BBS-2 treatment blocked NOS2 dimerization and completely inhibited the endotoxin-induced increase of plasma nitrate and nitrite levels. Treatment with BBS-2 after endotoxin administration prevented systemic hypotension and attenuated myocardial dysfunction. BBS-2 also prevented endotoxin-induced impairment of HPV. In contrast, treatment with NG-nitro-l-arginine methyl ester, which is an inhibitor of all three NOS isoforms, prevented the systemic hypotension but further aggravated the myocardial dysfunction associated with endotoxin challenge. Treatment with BBS-2 prevented endotoxin from causing key features of cardiovascular dysfunction in endotoxemic mice. Selective inhibition of NOS2 dimerization with BBS-2, while sparing the activities of other NOS isoforms, may prove to be a useful treatment strategy in sepsis.

Nitric oxide released from iNOS in polymorphonuclear leukocytes makes them deformable in an autocrine manner.

The objective of this study was to determine whether endogenous nitric oxide (NO) derived from reaction catalyzed by the inducible isoform of NO synthase (iNOS: NOS II) in polymorphonuclear leukocytes (PMNs) makes the PMNs deformable. Previous studies have shown that NO increases the deformability of PMNs and decreases the sequestration of PMNs in the lungs. However, there was little information regarding the effect of PMN-derived NO on the cells' deformability. In the present study PMNs were isolated from the blood of rats 24h after ip injection of saline (control) or lipopolysaccharide (LPS), and expression of iNOS in the PMNs of the LPS group was confirmed by immunocytochemistry. PMN deformability was evaluated by measuring the pressure generated during their passage through a microfilter at a constant flow rate. The nitrite/nitrate content of the solution in which the isolated PMNs were incubated was measured by the Griess method. In the control group, no iNOS was detectable in the PMNs, and the nitrite/nitrate level in the PMN incubation solution was low. Deformability was unchanged after incubation with specific iNOS inhibitor aminoguanidine, but decreased after incubation with N-formyl-methionyl-leucyl-phenyl-alanine. In the LPS group, PMN deformability was decreased compared to that of the control group. iNOS was detectable in the PMNs, and the deformability further decreased after incubation with aminoguanidine. These results suggest that endogenous NO generated during reactions catalyzed by iNOS in PMNs makes them deformable in an autocrine manner.

Myeloperoxidase-associated tyrosine nitration after intratracheal administration of lipopolysaccharide in rats.

BACKGROUND: Previous studies have shown that lipopolysaccharide-induced inflammation in the lung results in tyrosine nitration. The objective of this study was to evaluate the contribution of myeloperoxidase and peroxynitrite pathway to the tyrosine nitration in lipopolysaccharide-administered lungs of rats that were otherwise untreated or leukocyte-depleted by cyclophosphamide or received inhaled nitric oxide (NO). METHODS: The authors analyzed the immunoreactivity of inducible nitric oxide synthase (iNOS), nitrotyrosine (a product of the myeloperoxidase or peroxynitrite pathway), and chlorotyrosine (a byproduct of the myeloperoxidase pathway) by use of specific antibodies. The number of neutrophils in bronchoalveolar lavage fluid (BALF) and levels of myeloperoxidase activity in lung homogenates were also measured. RESULTS: Lipopolysaccharide enhanced the immunoreactivity of iNOS, nitrotyrosine, and chlorotyrosine in alveolar wall cells, alveolar macrophages, and neutrophils. Leukocyte depletion by cyclophosphamide and inhibition of leukocyte accumulation in the lungs by NO inhalation did not eliminate the increase in iNOS immunoreactivity in alveolar macrophages after lipopolysaccharide treatment, but nitrotyrosine and chlorotyrosine were not produced in these cells. Tyrosine nitration in response to lipopolysaccharide was associated with increases in neutrophil count in BALF and in myeloperoxidase activity in lung homogenates, whereas NO inhalation suppressed the neutrophil count in BALF and reduced tyrosine nitration and chlorination. CONCLUSIONS: These findings suggest that myeloperoxidase pathway has a role in tyrosine nitration in the lungs of lipopolysaccharide-treated rats, and that NO inhalation during early phase of inflammation does not increase but rather decreases tyrosine nitration and chlorination, possibly by reducing neutrophil sequestration.