Satoshi Homma

Activated neutrophils and platelet microaggregates impede blood filterability through microchannels during simulated extracorporeal circulation

BACKGROUND Neutrophil sequestration and platelet microaggregates in organ capillaries have been implicated in the inflammatory response associated with cardiopulmonary bypass. We examined the filterability of neutrophils and platelet microaggregates through silicon microchannels during simulated extracorporeal circulation. We hypothesize that blood contact with artificial surfaces over time decreases the ability of neutrophils, platelets, and their aggregates to pass through microchannels. METHODS Fresh human blood from donors (n = 9) was recirculated for 120 minutes in a simulated extracorporeal circuit. Blood samples were obtained from a donor at 0, 30, 60, and 120 minutes of recirculation. The microchannel transit time and the flow behavior of blood cells were evaluated by a silicon microchannel array flow analyzer. CD11b, L-selectin, and F-actin of neutrophils were measured by flow cytometry. Neutrophil and platelet counts and platelet aggregation to adenosine diphosphate were measured. RESULTS The microchannel transit time was prolonged during recirculation, reaching 185.9% +/- 25.6% of baseline at 120 minutes. The video microscope showed that neutrophils and platelet microaggregates plugged the microchannels. CD11b, L-selectin, and F-actin levels changed significantly by 120 minutes. Platelet counts decreased and platelet aggregability was attenuated. CONCLUSIONS Simulated extracorporeal circulation caused a progressive loss in the ability of neutrophils, platelets, and their aggregates to pass through the microchannels independent of neutrophil adhesion molecule expression.

Vasoconstrictor effects of endothelin-1 on myocardium microcirculation studied by the langendorff perfusion method: Differential sensitivities among microvessels ☆

An intravital fluorescence microscope system was used to investigate the pharmacological effects of endothelin-1 (ET-1) on the coronary microcirculation in the isolated beating hearts of rats. The heart was perfused by retrograde aortic steady flow with an oxygenated Krebs-Ringer solution containing FITC-dextran. Changes in diameters of coronary microvessels accompanying the cumulative injection of ET-1 in the perfusate were observed and recorded with a video camera system. Coronary perfusion pressure was also measured during each experiment. Bolus injections of ET-1 (1-300 pmole) elicited a dose-dependent increase in perfusion pressure from 54 +/- 6 mm Hg (mean +/- SEM; n = 10, before the ET-1 injection) to 144 +/- 9 mm Hg (n = 8, at the ET-1 dose of 300 pmole). A dose-dependent narrowing of microvessels was also observed. This vasoconstriction was especially prominent in small-sized arterioles; the maximum vasoconstriction of the smaller arterioles was significantly higher than that of the larger arterioles (P less than 0.05). The response induced by ET-1 dose of 3-10 pmole was significantly larger in arterioles than in postcapillary venules in the diameter range between 10 and 40 microns. The vasoconstriction produced by ET-1 was inhomogeneous. Some part of bifurcations of arterioles showed a prominent localized vasoconstriction, and occasionally showed a complete luminal obstruction. Such a segmental vasospasm might be attributed to localized sensitivities of arterioles to ET-1. These findings suggest that ET-1 may have an important role in governing the coronary resistance and regulating the capillary flow in the myocardium.

Effects of Endothelin-1 on Coronary Microcirculation in Isolated Beating Hearts of Rats

An intravital fluorescence videomicroscope system was used to investigate the pharmacological effects of endothelin-1 (ET-1) on the coronary microcirculation in isolated beating hearts of rats. The heart was perfused by retrograde aortic steady flow with an oxygenated Krebs-Ringer solution containing FITC-dextran. Cumulative injections of ET-1 (1-30 pmol) elicited a dose-dependent increase in perfusion pressure from 52 +/- 15 mm Hg (mean +/- SEM; n = 6) before the ET-1 injection to 104 +/- 23 mm Hg at the ET-1 dose of 30 pmol. A dose-dependent narrowing of microvessels was also observed on a monitor screen. This diffuse vasoconstriction was especially prominent in small arterioles; the maximum vasoconstriction of the smaller arterioles was significantly higher than that of the larger arterioles. These findings suggest that ET-1 may have an important role in governing the coronary resistance and regulating the capillary flow in myocardium.

ONO-6818, a novel, potent neutrophil elastase inhibitor, reduces inflammatory mediators during simulated extracorporeal circulation

BACKGROUND Among the serine proteases, neutrophil elastase is a powerful cytotoxic enzyme and plays a pivotal role in the inflammatory response associated with cardiopulmonary bypass. This study assesses the effects of the specific inhibition of neutrophil elastase by a novel, potent, low-molecular-weight neutrophil elastase inhibitor, ONO-6818. We hypothesized that ONO-6818 reduces inflammatory mediators and modulates adhesion molecules and the deformability of neutrophils during simulated extracorporeal circulation. METHODS Simulated extracorporeal circulation was established by recirculating fresh heparinized (3.75 U/mL) human blood for 120 minutes in a membrane oxygenator and a roller pump with and without 1.0 micromol/L of ONO-6818 (n = 9 for control group, n = 7 for ONO-6818 group). The neutrophil adhesion molecules, CD11b and L-selectin, and the cytoplasmic F-actin of neutrophils were measured by flow cytometry. Neutrophil deformability was evaluated using simulated silicon microcapillaries. Neutrophil elastase, interleukin 8, and C5b-9 were measured using enzyme immunoassay. RESULTS Neutrophil elastase levels were significantly lower in the ONO-6818 group. ONO-6818 significantly reduced interleukin 8 and C5b-9 production. ONO-6818 did not modulate changes of CD11b and L-selectin during recirculation. Cytoplasmic F-actin content and changes of neutrophil deformability did not significantly differ between the groups. CONCLUSIONS Inhibition of neutrophil elastase activity with ONO-6818 reduces further interleukin 8 production and the formation of the complement membrane attack complex, and this results in a reduction of neutrophil elastase levels during simulated extracorporeal circulation. This study suggests that specific neutrophil elastase inhibition with ONO-6818 is a feasible therapeutic option to attenuate the exaggerated inflammatory response associated with cardiopulmonary bypass.

Involvement of peptidyl-prolyl isomerase Pin1 in the inhibitory effect of fluvastatin on endothelin-1-induced cardiomyocyte hypertrophy

AIMS Cardiac hypertrophy is elicited by endothelin (ET)-1 as well as other neurohumoral factors, hemodynamic overload, and oxidative stress; HMG-CoA reductase inhibitors (statins) were shown to inhibit cardiac hypertrophy partly via the anti-oxidative stress. One of their common intracellular pathways is the phosphorylation cascade of MEK signaling. Pin1 specifically isomerizes the phosphorylated protein with Ser/Thr-Pro bonds and regulates their activity through conformational changes. There is no report whether the Pin1 activation contributes to ET-1-induced cardiomyocyte hypertrophy and whether the Pin1 inactivation contributes to the inhibitory effect of statins. The aim of this study was to reveal these questions. MAIN METHODS We assessed neonatal rat cardiomyocyte hypertrophy using ET-1 and fluvastatin by the cell surface area, ANP mRNA expression, JNK and c-Jun phosphorylation, and [(3)H]-leucine incorporation. KEY FINDINGS Fluvastatin inhibited ET-1-induced increase in the cell surface area, ANP expression, and [(3)H]-leucine incorporation; and it suppressed the signaling cascade from JNK to c-Jun. The phosphorylated Pin1 level, an inactive form, was decreased by ET-1; however, it reached basal level by fluvastatin. Furthermore, Pin1 overexpression clearly elicited cardiomyocyte hypertrophy, which was inhibited by fluvastatin. SIGNIFICANCE This is the first report that ET-1-induced cardiomyocyte hypertrophy is mediated through the Pin1 activation and that the inhibitory effect of fluvastatin on cardiomyocyte hypertrophy would partly be attributed to the suppression of the Pin1 function. This study firstly suggests that Pin1 determines the size of hypertrophied cardiomyocyte by regulating the activity of phosphorylated molecules and that statins exert their pleiotropic effects partly via Pin1 inactivation.

Calcitonin gene-related peptide protects the myocardium from ischemia induced by endothelin-1: Intravital microscopic observation and 31P-MR spectroscopic studies☆

AIMS Calcitonin gene-related peptide (CGRP) is a potent vasodilator neuropeptide. We investigated the ameliorating effect of CGRP in myocardial ischemia induced by endothelin-1 (ET-1), with special emphasis on myocardial microvascular hemodynamics and levels of energy-related metabolites. MAIN METHODS The Langendorff preparations of rat isolated heart were perfused at a constant flow rate. Microvascular blood flow was also visualized in the anterior epicardium of the left ventricle by means of an intravital fluorescence microscope system. Energy-related metabolite contents in the myocardium were measured by means of (31)P-magnetic resonance spectroscopy ((31)P-MRS). KEY FINDINGS Intracoronary bolus injections of CGRP caused dose-dependent decreases in coronary perfusion pressure (CPP) in the hearts exposed to ET-1 (30 pmol). The vasodilator potency of CGRP was about 10,000-fold greater than that of nitroglycerin and 1,000-fold greater than that of isobutylmethylxanthine. Vasodilation of the small-sized arterioles (10-40 μm in diameter) in response to CGRP (100 pmol) was confirmed by direct microscopic observation. After ET-1 (30 pmol) plus vehicle administration, high energy phosphates (phosphocreatine (PCr), ATP) were markedly reduced (p<0.05). CGRP administration significantly (p<0.05) attenuated the anaerobic changes in the myocardium (decrease in PCr) and macrohemodynamic alterations (increase in CPP, decrease in dP/dt etc.) induced by ET-1. SIGNIFICANCE We conclude that CGRP effectively confers hemodynamic and metabolic protections to isolated beating hearts against ET-1-induced myocardial ischemia.

Effects of selective endothelin (ET)-A receptor antagonist versus dual ET-A/B receptor antagonist on hearts of streptozotocin-treated diabetic rats

AIMS The aim was to study the differences in the effectiveness of two types of endothelin (ET) receptor antagonists (selective ET-A or dual ET-A/B antagonists) on the hearts of streptozotocin (STZ)-induced diabetic rats (type I diabetes) at functional and biochemical/molecular levels. MAIN METHODS Citrate saline (vehicle) or STZ was injected into rats. The ET-A/B dual receptor antagonist (SB209670, 1mg/kg/day) and the ET-A receptor antagonist (TA-0201, 1mg/kg/day) were then administered to these rats. One week after injection, the animals were separated into those receiving SB209670, TA-0201 or vehicle by 4-week osmotic mini-pump. KEY FINDINGS The VEGF level and percent fractional shortening in the diabetic heart were significantly decreased compared to the non-diabetic heart, whereas SB209670 and TA-0201 treatments greatly and comparably prevented this decrease. SB209670 treatment was more effective in reversing decreased expressions of KDR and phosphorylated AKT, downstream of VEGF angiogenic signaling, than TA-0201 treatment. The eNOS levels in hearts were significantly higher in diabetic rats than in healthy rats, and this increase was significantly reduced by TA-0210 but not by SB209670 treatment. SIGNIFICANCE Improvement of KDR mRNA and pAKT levels by SB209670 but not TA-0201 suggests that dual ET-A/-B blockade may be effective in improving intracellular systems of these components in the diabetic rat heart. However, the present study also showed that TA-0201 or SB209670 improved percent fractional shortening and VEGF levels of the diabetic hearts to a similar extent, suggesting that ET-A blockade and dual ET-A/-B blockade are similarly effective in improving cardiac dysfunction in the diabetic rats.

Endothelin-1-induced cardiomyocyte hypertrophy is partly regulated by transcription factor II-F interacting C-terminal domain phosphatase of RNA polymerase II.

AIMS Cardiac hypertrophy is associated with the increase of total amount of RNA, which is in accordance with RNA polymerase II (RNAPII) activation via C-terminal domain (CTD) phosphorylation of the largest subunit of RNAPII. It has been demonstrated that endothelin-1 (ET-1) phosphorylates CTD at the hypertrophic response in cardiomyocytes. However, it is unclear whether ET-1-induced hypertrophy is affected by the CTD phosphatase, transcription factor IIF-interacting CTD phosphatase1 (FCP1). MAIN METHODS We analyzed whether ET-1-induced cardiomyocyte hypertrophy was affected by overexpression of FCP1 or dominant-negative form of FCP1 (dnFCP1) in neonatal rat cardiomyocytes. KEY FINDINGS The level of ET-1-induced RNAPII CTD phosphorylation was decreased by FCP1 overexpression, whereas it was sustained by dnFCP1. Global RNA synthesis evaluated by [(3)H]-uridine incorporation showed that the ET-1-induced increase in RNA synthesis was suppressed by FCP1 and was augmented by dnFCP1. ET-1-induced increase in cell surface area was suppressed by FCP1 and was preserved by dnFCP1. Furthermore, the ET-1-induced increase in molecular markers of cardiac hypertrophy, expression of ANP and β-MHC gene, was suppressed by FCP1 and was not inhibited by dnFCP1. SIGNIFICANCE ET-1-induced cardiac hypertrophy and CTD phosphorylation level are functionally regulated by FCP1. These findings suggest that FCP1 plays an important role in ET-1-induced cardiac hypertrophy via controlling phosphorylation level of the RNAPII CTD.

Flow behavior of erythrocytes in living microvessels: Analysis of the distribution of dynamic hematocrits measured in vivo

Abstract We developed a new method for visualizing the flow behavior of erythrocytes in living microvessels by the fluorescent labeling technique. Intravital microscopic observation of the microcirculation in the rat mesentery using this technique enabled quantitative analyses of a few hydrodynamic aspects of the two-phase blood flow; e.g. diameter-flow relationships in a consecutively branched vascular network and the distribution of erythrocytes in an arteriolar bifurcation.