Novel Synthesized Radical-Containing Nanoparticles Limit Infarct Size Following Ischemia and Reperfusion in Canine Hearts.

PURPOSE: Although nitroxyl radicals such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) scavenge free radicals, their short half-life and considerable side effects such as systemic hypotension and bradycardia have limited their clinical application. Since a radical-containing nanoparticle (RNP) delivers nitroxyl radicals with a prolonged half-life specific to ischemic hearts, we investigated whether RNPs reduce infarct size without the occurrence of substantial side effects and whether nitric oxide (NO) contributes to the cardioprotective effects of RNPs. METHODS: The left anterior descending coronary arteries of dogs were occluded for 90 min, followed by reperfusion for 6 h. Either RNPs, micelles (not containing TEMPO) (control), or 4-hydroxy-TEMPO (TEMPOL) was injected into a systemic vein for 5 min before reperfusion. We evaluated the infarct size, myocardial apoptosis, plasma NO levels in coronary venous blood, and the RNP spectra using an electron paramagnetic resonance assay. RESULTS: RNPs reduced infarct size compared with the control group and TEMPOL group (19.5 ± 3.3 vs. 42.2 ± 3.7 vs. 30.2 ± 3.4%). RNPs also reduced myocardial apoptosis compared with the control and TEMPOL group. Coronary venous NO levels increased in the RNP group. CONCLUSIONS: In conclusion, the administration of 2,2,6,6-tetramethylpiperidine-1-oxyl as a RNP exerted cardioprotective effects against ischemia and reperfusion injury in canine hearts without exerting unfavorable hemodynamic effects. RNPs may represent a promising new therapy for patients with acute myocardial infarction.

Visualization of microvessels by angiography using inverse-Compton scattering X-rays in animal models.

The fundamental performance of microangiography has been evaluated using the S-band linac-based inverse-Compton scattering X-ray (iCSX) method to determine how many photons would be required to apply iCSX to human microangiography. ICSX is characterized by its quasi-monochromatic nature and small focus size which are fundamental requirements for microangiography. However, the current iCSX source does not have sufficient flux for microangiography in clinical settings. It was determined whether S-band compact linac-based iCSX can visualize small vessels of excised animal organs, and the amount of X-ray photons required for real time microangiography in clinical settings was estimated. The iCSX coupled with a high-gain avalanche rushing amorphous photoconductor camera could visualize a resolution chart with only a single iCSX pulse of ∼3 ps duration; the resolution was estimated to be ∼500 µm. The iCSX coupled with an X-ray cooled charge-coupled device image sensor camera visualized seventh-order vascular branches (80 µm in diameter) of a rabbit ear by accumulating the images for 5 and 30 min, corresponding to irradiation of 3000 and 18000 iCSX pulses, respectively. The S-band linac-based iCSX visualized microvessels by accumulating the images. An iCSX source with a photon number of 3.6 × 10(3)-5.4 × 10(4) times greater than that used in this study may enable visualizing microvessels of human fingertips even in clinical settings.

H(2) mediates cardioprotection via involvements of K(ATP) channels and permeability transition pores of mitochondria in dogs.

PURPOSE: Inhalation of hydrogen (H(2)) gas has been shown to limit infarct size following ischemia-reperfusion injury in rat hearts. However, H(2) gas-induced cardioprotection has not been tested in large animals and the precise cellular mechanism of protection has not been elucidated. We investigated whether opening of mitochondrial ATP-sensitive K+ channels (mK(ATP)) and subsequent inhibition of mitochondrial permeability transition pores (mPTP) mediates the infarct size-limiting effect of H(2) gas in canine hearts. METHODS: The left anterior descending coronary artery of beagle dogs was occluded for 90 min followed by reperfusion for 6 h. Either 1.3% H(2) or control gas was inhaled from 10 min prior to start of reperfusion until 1 h of reperfusion, in the presence or absence of either 5-hydroxydecanoate (5-HD; a selective mK(ATP) blocker), or atractyloside (Atr; a mPTP opener). RESULTS: Systemic hemodynamic parameters did not differ among the groups. Nevertheless, H(2) gas inhalation reduced infarct size normalized by risk area (20.6±2.8% vs. control gas 44.0±2.0%; p<0.001), and administration of either 5-HD or Atr abolished the infarct size-limiting effect of H(2) gas (42.0±2.2% with 5-HD and 45.1±2.7% with Atr; both p<0.001 vs. H(2) group). Neither Atr nor 5-HD affected infarct size per se. Among all groups, NAD content and the number of apoptotic and 8-OHdG positive cells was not significantly different, indicating that the cardioprotection afforded by H(2) was not due to anti-oxidative actions or effects on the NADH dehydrogenase pathway. CONCLUSIONS: Inhalation of H(2) gas reduces infarct size in canine hearts via opening of mitochondrial K(ATP) channels followed by inhibition of mPTP. H(2) gas may provide an effective adjunct strategy in patients with acute myocardial infarction receiving reperfusion therapy.

A rotating cerium anode X-ray system allows visualization of intramural coronary vessels after cardiac stem cell therapy for myocardial infarction.

Conventional angiography is insufficient for evaluating the therapeutic effect of cardiac regeneration therapy. A microangiographic X-ray system using a cerium anode was developed. Cerium has a characteristic X-ray with a peak at 34.6 keV, which allows visualization of tiny amounts of iodine. The performance of the cerium anode X-ray system was evaluated in two excised normal canine hearts and in excised ischemic canine hearts treated with c-kit-positive cardiac stem cells (5 canines) or without cells (5 control canines). In the normal canines, branches penetrating from the left anterior descending artery into the myocardium were visualized, down to third-order branches. In just the treated hearts treated with stem cells, small vessels characterized by irregular vessel walls were observed. The cerium anode X-ray system allowed visualization of microvessels in excised ischemic canine hearts, and may evaluate the effect of cardiac stem cell therapy.

Evaluation of Intracranial Microvessel Visualization in Mouse and Dog Models by Using a New Rotating Cerium Anode X-ray System.

OBJECTIVE: Lacunar stroke may be caused by infarction of small perforating branches of the middle cerebral artery. We developed a microangiographic X-ray system using a cerium anode to evaluate the perforating branches. METHODS: Iodine has K-edges at 33.2 kilo electron volts. Cerium yields a characteristic X-ray of 34.6 kilo electron volts, therefore, the cerium anode X-ray system could detect tiny amounts of contrast material. First, an X-ray chart was used to evaluate the resolution. Second, the brains of mice were dissected and irradiated. Third, the brains of dogs were excluded and irradiated. Fourth, iodine was perfused into the carotid artery of living dogs during brain imaging. RESULTS: In the first experiment, the cerium anode X-ray system elicited 4.86 clear line pairs. In mice, the perforating branches of the middle cerebral artery could be visualized. The perforating branches were clearly observed in dog brains ex situ even through an acrylic plate, but not in conventional X-ray images. Iodine moving inside the perforating branches was visualized in dog brains in situ using the cerium anode X-ray system. CONCLUSION: The cerium anode X-ray system allowed us to visualize the perforating branches of the middle cerebral artery in living dogs.

Beneficial effect of hydroxyfasudil, a specific Rho-kinase inhibitor, on ischemia/reperfusion injury in canine coronary microcirculation in vivo.

OBJECTIVES: We examined whether hydroxyfasudil, a specific Rho-kinase inhibitor, exerts cardioprotective effect on coronary ischemia/reperfusion (I/R) injury and, if so, whether nitric oxide (NO) is involved. BACKGROUND: Recent studies have demonstrated that Rho-kinase is substantially involved in the pathogenesis of cardiovascular diseases; however, it remains to be examined whether it is also involved in ischemia/reperfusion (I/R) injury. METHODS: Canine subepicardial small arteries (SA, >or=100 microm) and arterioles (A, <100 microm) were observed by a charge-coupled device intravital microscope during I/R. Coronary vascular responses to endothelium-dependent (acetylcholine, intracoronary [IC]) and -independent (papaverine, IC) vasodilators were examined after I/R under the following four conditions: control (n = 7), NO synthase inhibitor alone (N(G)-monomethl-L-arginine [L-NMMA], IC, n = 4), hydroxyfasudil alone (IC, n = 7), and hydroxyfasudil plus L-NMMA (n = 7). RESULTS: Hydroxyfasudil significantly attenuated serotonin (IC)-induced vasoconstriction of SA (-7 +/- 1% vs. 2 +/- 1%, p < 0.01). Coronary I/R significantly impaired coronary vasodilation to acetylcholine after I/R (SA, p < 0.05; and A, p < 0.01 vs. before I/R) and L-NMMA further reduced the vasodilation, whereas hydroxyfasudil completely preserved the responses. The vasoconstriction by L-NMMA after I/R was significantly improved by hydroxyfasudil in both-sized arteries (both p < 0.01). Expression of endothelial nitric oxide synthase (eNOS) protein in the ischemic endocardium of left anterior descending coronary artery area (as determined by Western blotting) significantly decreased (79 +/- 4%) compared with the nonischemic endocardium of LCX area (100 +/- 7%), which was improved by hydroxyfasudil (105 +/- 6%, p < 0.01). Hydroxyfasudil significantly reduced myocardial infarct size, and hydroxyfasudil with L-NMMA also reduced the infarct size compared with L-NMMA alone. CONCLUSIONS: Hydroxyfasudil exerts cardioprotective effects on coronary I/R injury in vivo, in which NO-mediated mechanism may be involved through preservation of eNOS expression.

Triiodothyronine acutely increases blood flow in the ventricles and kidneys of anesthesized rabbits.

Thyroid hormone (triiodothyronine [T(3)]) has various nongenomic effects, including alterations in glucose and fatty acid metabolism, augmentation of intracellular Ca(2+), enhancement of myocardial contractility, and vascular dilatation. However, its effect on regional blood flow remains to be established. We have measured the effect of T(3) on blood flow in major organs of anesthetized rabbits in vivo using the microsphere method. Under artificial respiration, nonradioactive microspheres (5 x 10(5)) labeled with barium were injected to measure blood flow at control level. Then, T(3) (50 microg/kg per milliliter) was administered and microspheres labeled with iodine (5 x 10(5)) were injected. The atria, ventricles, kidneys, and right upper limb were excised and their contents of microspheres were evaluated. Blood flow in the ventricles was significantly increased by T(3) (2.9 +/- 0.3 versus 3.4 +/- 0.3 mL/min per gram, vehicle versus T(3)). Similarly, blood flow in the kidneys was significantly higher after T(3) injection (4.3 +/- 0.5 versus 5.1 +/- 0.5 mL/min per, vehicle versus T(3)). The blood flow in the atria and skeletal muscles remained unchanged. These results indicate that the vasodilatory response to T(3) is not uniform and occurs preferentially in major organs such as cardiac ventricles and kidneys; this may be relevant to the T(3)-induced improvement of cardiac function.

Optimal windows of statin use for immediate infarct limitation: 5'-nucleotidase as another downstream molecule of phosphatidylinositol 3-kinase.

BACKGROUND: Although statins are reported to have a cardioprotective effect, their immediate direct influence on ischemia-reperfusion injury and the underlying mechanisms remain obscure. We investigated these issues an in vivo canine model. METHODS AND RESULTS: Dogs were subjected to coronary occlusion (90 minutes) and reperfusion (6 hours) immediately after injection of pravastatin (0.2, 2, or 10 mg/kg), pitavastatin (0.01, 0.1, or 0.5 mg/kg), or cerivastatin (0.5, 5, or 50 microg/kg). Then myocardial phosphatidylinositol 3-kinase (PI3-K) and 5'-nucleotidase activities were measured, as well as infarct size. After 15 minutes of reperfusion, pravastatin caused dose-dependent activation of Akt and ecto-5'-nucleotidase in the ischemic zone, and the effect was significant at higher doses. Pitavastatin also significantly increased these activities, and its optimal dose was within the clinical range, whereas cerivastatin caused activation at the lowest dose tested. In all cases, both Akt and ecto-5'-nucleotidase showed activation in parallel, and this activation was completely abolished by wortmannin, a PI3-K inhibitor. The magnitude of the infarct-limiting effect paralleled the increase in Akt and ecto-5'-nucleotidase activity and was blunted by administration of wortmannin, alpha,beta-methyleneadenosine-5'-diphosphate, or 8-sulfophenyltheophylline during reperfusion. Both collateral flow and the area at risk were comparable for all groups. CONCLUSIONS: Activation of ecto-5'-nucleotidase after ischemia by PI3-K activation may be crucial for immediate infarct-size limitation by statins. There seems to be an optimal dose for each statin that is independent of its clinical cholesterol-lowering effect.

Protein kinase A as another mediator of ischemic preconditioning independent of protein kinase C.

BACKGROUND: We and others have reported that transient accumulation of cyclic AMP (cAMP) in the myocardium during ischemic preconditioning (IP) limits infarct size independent of protein kinase C (PKC). Accumulation of cAMP activates protein kinase A (PKA), which has been demonstrated to cause reversible inhibition of RhoA and Rho-kinase. We investigated the involvement of PKA and Rho-kinase in the infarct limitation by IP. METHODS AND RESULTS: Dogs were subjected to 90-minute ischemia and 6-hour reperfusion. We examined the effect on Rho-kinase activity during sustained ischemia and infarct size of (1) preischemic transient coronary occlusion (IP), (2) preischemic activation of PKA/PKC, (3) inhibition of PKA/PKC during IP, and (4) inhibition of Rho-kinase or actin cytoskeletal deactivation during myocardial ischemia. Either IP or dibutyryl-cAMP treatment activated PKA, which was dose-dependently inhibited by 2 PKA inhibitors (H89 and Rp-cAMP). IP and preischemic PKA activation substantially reduced infarct size, which was blunted by preischemic PKA inhibition. IP and preischemic PKA activation, but not PKC activation, caused a substantial decrease of Rho-kinase activation during sustained ischemia. These changes were cancelled by preischemic inhibition of PKA but not PKC. Furthermore, either Rho-kinase inhibition (hydroxyfasudil or Y27632) or actin cytoskeletal deactivation (cytochalasin-D) during sustained ischemia achieved the same infarct limitation as preischemic PKA activation without affecting systemic hemodynamic parameters, the area at risk, or collateral blood flow. CONCLUSIONS: Transient preischemic activation of PKA reduces infarct size through Rho-kinase inhibition and actin cytoskeletal deactivation during sustained ischemia, implicating a novel mechanism for cardioprotection by ischemic preconditioning independent of PKC and a potential new therapeutic target.

Beta-adrenoceptor blocker carvedilol provides cardioprotection via an adenosine-dependent mechanism in ischemic canine hearts.

BACKGROUND: Carvedilol is a beta-adrenoceptor blocker with a vasodilatory action that is more effective for the treatment of congestive heart failure than other beta-blockers. Recently, carvedilol has been reported to reduce oxidative stress, which may consequently reduce the deactivation of adenosine-producing enzymes and increase cardiac adenosine levels. Therefore, carvedilol may also have a protective effect on ischemia and reperfusion injury, because adenosine mediates cardioprotection in ischemic hearts. METHODS AND RESULTS: In anesthetized dogs, the left anterior descending coronary artery was occluded for 90 minutes, followed by reperfusion for 6 hours. Carvedilol reduced the infarct size (15.0+/-2.8% versus 40.9+/-4.2% in controls), and this effect was completely reversed by the nonselective adenosine receptor antagonist 8-sulfophenyltheophylline (45.2+/-5.4%) or by an inhibitor of ecto-5'-nucleotidase (44.4+/-3.6%). There were no differences of either area at risk or collateral flow among the various groups. When the coronary perfusion pressure was reduced in other dogs so that coronary blood flow was decreased to 50% of the nonischemic level, carvedilol increased coronary blood flow (49.4+/-5.6 to 73.5+/-7.5 mL x 100 g(-1) x min(-1); P<0.05) and adenosine release (112.3+/-22.2 to 240.6+/-57.1 nmol/L; P<0.05) during coronary hypoperfusion. This increase of coronary blood flow was attenuated by either 8-sulfophenyltheophylline or superoxide dismutase. In human umbilical vein endothelial cells cultured with or without xanthine and xanthine oxidase, carvedilol caused an increase of ecto-5'-nucleotidase activity. CONCLUSIONS: Carvedilol shows a cardioprotective effect against ischemia and/or reperfusion injury via adenosine-dependent mechanisms.

Amelioration of ischemia- and reperfusion-induced myocardial injury by the selective estrogen receptor modulator, raloxifene, in the canine heart.

OBJECTIVES: We sought to investigate whether raloxifene reduces ischemia-reperfusion injury and what mechanisms are involved in the cardioprotective effects. BACKGROUND: Estradiol-17-beta reduces myocardial infarct size in ischemia-reperfusion injury. Raloxifene, a selective estrogen receptor modulator, demonstrates immediate coronary artery vasorelaxing effects. METHODS: The myocardial ischemia-reperfusion model included anesthetized open-chest dogs after 90-min occlusion of the left anterior descending coronary artery (LAD) and subsequent 6-h reperfusion. Raloxifene and/or other drugs were infused into the LAD from 10 min before coronary occlusion to 1 h after reperfusion without an occlusion period. RESULTS: Infarct size was reduced in the raloxifene (5 microg/kg per min) group compared with the control group (7.2 +/- 2.5% vs. 40.9 +/- 3.9% of the area at risk, p < 0.01). Either N(G)-nitro-L-arginine methyl ester (L-NAME), the inhibitor of nitric oxide (NO) synthase, or charybdotoxin, the blocker of Ca(2+)-activated K+ (K(Ca)) channels, partially attenuated the infarct size-limiting effect, and both of them completely abolished the effect. The incidence of ventricular fibrillation was also less in the raloxifene group than in the control group (11% vs. 44%, p < 0.05). Activity of p38 mitogen-activated protein (MAP) kinase increased with 15-min ischemia, and raloxifene pretreatment inhibited the activity. Myeloperoxidase activity of the 6-h reperfused myocardium was also attenuated by raloxifene. CONCLUSIONS: These data demonstrate that raloxifene reduces myocardial ischemia-reperfusion injury by mechanisms dependent on NO and the opening of K(Ca) channels in canine hearts. Deactivation of p38 MAP kinase and myeloperoxidase by raloxifene may be involved in the cellular mechanisms of cardioprotection.

Biodegradable gelatin hydrogel potentiates the angiogenic effect of fibroblast growth factor 4 plasmid in rabbit hindlimb ischemia.

OBJECTIVES: We investigated the potentiation of gene therapy using fibroblast growth factor 4 (FGF4)-gene by combining plasmid deoxyribonucleic acid (DNA) with biodegradable gelatin hydrogel (GHG). BACKGROUND: Virus vectors transfer genes efficiently but are biohazardous, whereas naked DNA is safer but less efficient. Deoxyribonucleic acid charges negatively; GHG has a positively charged structure and is biodegradable and implantable; FGF4 has an angiogenic ability. METHODS: The GHG-DNA complex was injected into the hindlimb muscle (63 mice and 55 rabbits). Gene degradation was evaluated by using (125)I-labeled GHG-DNA complex in mice. Transfection efficiency was evaluated with reverse-transcription nested polymerase chain reaction and X-Gal histostaining. The therapeutic effects of GHG-FGF4-gene complex (GHG-FGF4) were evaluated in rabbits with hindlimb ischemia. RESULTS: Gelatin hydrogel maintained plasmid in its structure, extending gene degradation temporally until 28 days after intramuscular delivery, and improving transfection efficiency. Four weeks after gene transfer, hindlimb muscle necrosis was ameliorated more markedly in the GHG-FGF4 group than in the naked FGF4-gene and GHG-beta-galactosidase (control) groups (p < 0.05, Kruskal-Wallis test). Synchrotron radiation microangiography (spatial resolution, 20 microm) and flow determination with microspheres confirmed significant vascular responsiveness to adenosine administration in the GHG-FGF4 group, but not in the naked FGF4-gene and the control. CONCLUSIONS: The GHG-FGF4 complex promoted angiogenesis and blood flow regulation of the newly developed vessels possibly by extending gene degradation and improving transfection efficiency without the biohazard associated with viral vectors.

Development of Fingertip Synchrotron Radiation Microangiography toward Clinical Prediction of Diabetic Microangiopathy.

OBJECTIVES: The spatial resolution of conventional angiographic systems is not enough to predict diabetic microangiopathy in arterioles (20-200 µm). METHODS: To determine whether fingertip synchrotron (SR) radiation microangiography has enough spatial resolution to quantitate arteriolar diameter changes, and whether an arteriolar paradoxical vasoconstriction is a characteristic observation for diabetic microangiopathy, diameter reduction as arteriolar branching and difference of the diameter changes induced by acetylcholine between control (n = 5) and diabetic rats (n = 5) were analyzed. RESULTS: Fingertip SR microangiography visualized the arterioles with a diameter range of 30-300 µm and demonstrated vascular diameter reduction as branching with a fixed ratio (r = 0.93, P < 0.004 and r = 0.73, P < 0.001). A vasodilatory reaction was induced by acetylcholine in the control (142.4 ± 61.9 to 190.9 ± 73.5, P < 0.05, n = 25), in contrast, paradoxical vasoconstriction in diabetic rats (201.6 ± 83.0 to 16 0.4 ± 67.9, P < 0.05, n = 37). Histological angiopathy was noted only in the diabetic rats. CONCLUSION: In conclusion, the fingertip SR microangiography is useful to predict diabetic micrangiopahty.

Altered blood flow in cerebral perforating arteries of rat models of diabetes: A synchrotron radiation microangiographic study toward clinical evaluation of white matter hyperintensities.

AIM: As altered blood flow in the cerebral perforating arteries (PA) might be related to development of cerebral white matter hyperintensities, we examined whether the hemodynamic relationship of the PA and middle cerebral artery (MCA) is altered in rat models of diabetes, compared with normal rats and a rat model of sinoatrial denervation (blood pressure fluctuation model). METHODS: We used microangiography with monochromatic synchrotron radiation to measure the diameters of the PA and MCA at 4.5 µm resolution in five groups of rats: (i) Long-Evans Tokushima Otsuka (LETO); (ii) Otsuka Long-Evans Tokushima Fatty (a model of type 2 diabetes with obesity); (iii) LETO with sinoaortic denervation (LETO + SAD); (iv) F344; and (v) F344 + streptozotocin (a model of type 1 diabetes). RESULTS: Compared with LETO, Otsuka Long-Evans Tokushima Fatty rats showed a significant reduction in the diameter of both PA and MCA, though the PA/MCA diameter ratio was unchanged. In contrast, compared with LETO, LETO + SAD rats showed an increased MCA diameter, and the PA/MCA diameter ratio was decreased. Compared with F344 rats, the MCA diameter was increased in F344 + streptozotocin rats, and the PA/MCA diameter ratio was decreased. Scatter diagrams showed that the diameters of the PA and MCA were essentially independent of each other in the two types of diabetic models. CONCLUSION: PA were consistently visualized at high resolution by means of microangiography using synchrotron radiation. The present results show that rat diabetic models exhibit changes in PA diameter and PA/MCA diameter ratio, which might be related to the development of diabetes-associated cerebral white matter hyperintensities.

Inhomogeneous vasodilatory responses of rat tail arteries to heat stress: evaluation by synchrotron radiation microangiography.

Tail blood flow is crucial for dissipating body heat in rats. Angiographies are convenient tools to evaluate tail circulation. However, conventional angiographies do not have sufficient sensitivity or spatial resolution for small vessels. Recently, we developed a novel microangiographic system using monochromatic synchrotron radiation and a high-definition video camera system. Here, we report an evaluation of rat tail circulation under heat stress using the synchrotron radiation microangiographic system. We performed an experiment using the microangiography of the caudal artery before and after heating up WKAH/HkmSlc rats to rectal temperature of 39 degrees C. The images were digitized and temporal subtraction was performed, and the diameters of caudal arteries were evaluated. After heating, the medial caudal artery was markedly dilated (320 +/- 53 to 853 +/- 243 micro m in diameter, p<0.001), while no significant change was observed in the lateral caudal arteries (139 +/- 42 to 167 +/- 73 micro m) and segmental anastomosing vessels. The heat stress allowed for visualization of the superficial caudal arteries with a diameter of approximately 60 micro m, not visible prior to heating. Thus, synchrotron radiation microangiography demonstrated that the rat tail possessed dual sets of arteries; one set was highly sensitive to heat-induced vasodilation (medial caudal artery and superficial caudal arteries) and the other set was less sensitive (lateral caudal arteries and segmental anastomosing vessels).

Carperitide induces coronary vasodilation and limits infarct size in canine ischemic hearts: role of NO.

Carperitide is effective for heart failure (HF) owing to its diuretic and vasodilatory effects. This recombinant peptide may also have direct cardioprotective effects because carperitide reduces the severity of heart failure and limits infarct size. Because coronary vasodilation is an important cardioprotective treatment modality, we investigated whether carperitide increased coronary blood flow (CBF) and improved myocardial metabolic and contractile dysfunction during ischemia in canine hearts. We also tested whether carperitide is directly responsible for limiting the infarct size. We infused carperitide at 0.025-0.2 µg kg(-1) min(-1) into the canine coronary artery. A minimum dose of 0.1 µg kg(-1) min(-1) was required to obtain maximal vasodilation. To test the effects of carperitide on ischemic hearts, we reduced perfusion pressure in the left anterior descending coronary artery such that CBF decreased to one-third of the baseline value. At 10 min after carperitide was infused at a dose of 0.1 µg kg(-1) min(-1), we observed increases in CBF, fractional shortening (FS) and pH levels in coronary venous blood without concomitant increases in cardiac nitric oxide (NO) levels; these changes were attenuated using either the atrial natriuretic peptide receptor antagonist HS-142-1 or the NO synthase inhibitor L(ω)-nitroarginine methyl ester (L-NAME). Cyclic guanosine monophosphate (GMP) levels in the coronary artery were elevated in response to carperitide that also limited the infarct size after 90 min of ischemia and subsequent reperfusion. Again, these effects were blunted by L-NAME. Carperitide increases CBF, reduces myocardial contractile and metabolic dysfunction and limits infarct size. In addition, NO is necessary for carperitide-induced vasodilation and cardioprotection in ischemic hearts.

Role of Cu,Zn-SOD in the synthesis of endogenous vasodilator hydrogen peroxide during reactive hyperemia in mouse mesenteric microcirculation in vivo.

We have recently demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor and that endothelial Cu/Zn-superoxide dismutase (SOD) plays an important role in the synthesis of endogenous H2O2 in both animals and humans. We examined whether SOD plays a role in the synthesis of endogenous H2O2 during in vivo reactive hyperemia (RH), an important regulatory mechanism. Mesenteric arterioles from wild-type and Cu,Zn-SOD(-/-) mice were continuously observed by a pencil-type charge-coupled device (CCD) intravital microscope during RH (reperfusion after 20 and 60 s of mesenteric artery occlusion) in the cyclooxygenase blockade under the following four conditions: control, catalase alone, N(G)-monomethyl-L-arginine (L-NMMA) alone, and L-NMMA + catalase. Vasodilatation during RH was significantly decreased by catalase or L-NMMA alone and was almost completely inhibited by L-NMMA + catalase in wild-type mice, whereas it was inhibited by L-NMMA and L-NMMA + catalase in the Cu,Zn-SOD(-/-) mice. RH-induced increase in blood flow after L-NMMA was significantly increased in the wild-type mice, whereas it was significantly reduced in the Cu,Zn-SOD(-/-) mice. In mesenteric arterioles of the Cu,Zn-SOD(-/-) mice, Tempol, an SOD mimetic, significantly increased the ACh-induced vasodilatation, and the enhancing effect of Tempol was decreased by catalase. Vascular H(2)O(2) production by fluorescent microscopy in mesenteric arterioles after RH was significantly increased in response to ACh in wild-type mice but markedly impaired in Cu,Zn-SOD(-/-) mice. Endothelial Cu,Zn-SOD plays an important role in the synthesis of endogenous H(2)O(2) that contributes to RH in mouse mesenteric smaller arterioles.

Effect of systemic blood pressure on microcollateral circulation evaluated by real-time contrast echocardiography.

BACKGROUND: In acute myocardial infarction, residual collateral-derived myocardial blood flow (CBF) within the ischemic area is one of the major determinants of infarct size. Management of systemic blood pressure (sBP) related to maintain collateral circulation is still difficult. The aim of this study was to reveal the influence of sBP on the rescue of area at risk by collateral circulation. METHODS: Real-time myocardial contrast echocardiography just after the onset of complete occlusion of the left circumflex coronary artery was performed in collateral-rich open-chest dogs. The video intensity of the ischemic area was evaluated during the occlusion and the CBF (A x beta) was calculated from a replenishment curve: y = A (1 - e(-beta t)). To analyze the effect of sBP on the collateral circulation, sBP was altered by infusion of nitroglycerin or etilefrine hydrochloride. To evaluate the defect size (%DS), every end-systolic myocardial contrast echocardiography image after left circumflex coronary artery occlusion was converted into binary images using custom offline software. RESULTS: The %DS increased and CBF slightly decreased at low sBP. The %DS decreased and CBF increased at high sBP. At excessively high sBP, %DS increased and CBF decreased again. CONCLUSION: Real-time myocardial contrast echocardiography, which is a useful noninvasive method to evaluate the collateral perfusion quantitatively, has a crucial role in the decision of patient treatment and management strategy of acute myocardial infarction.

Important role of endogenous hydrogen peroxide in pacing-induced metabolic coronary vasodilation in dogs in vivo.

OBJECTIVES: We examined whether endogenous hydrogen peroxide (H2O2) is involved in pacing-induced metabolic vasodilation in vivo. BACKGROUND: We have previously demonstrated that endothelium-derived H2O2 is an endothelium-derived hyperpolarizing factor in canine coronary microcirculation in vivo. However, the role of endogenous H2O2 in metabolic coronary vasodilation in vivo remains to be examined. METHODS: Canine subepicardial small coronary arteries (> or =100 microm) and arterioles (<100 microm) were continuously observed by a microscope under cyclooxygenase blockade (ibuprofen, 12.5 mg/kg intravenous [IV]) (n = 60). Experiments were performed during paired right ventricular pacing under the following 7 conditions: control, nitric oxide (NO) synthase inhibitor (N(G)-monomethyl-L-arginine [L-NMMA], 2 micromol/min for 20 min intracoronary [IC]), catalase (a decomposer of H2O2, 40,000 U/kg IV and 240,000 U/kg/min for 10 min IC), 8-sulfophenyltheophylline (SPT) (an adenosine receptor blocker, 25 mug/kg/min for 5 min IC), L-NMMA+catalase, L-NMMA+tetraethylammonium (TEA) (K(Ca)-channel blocker, 10 microg/kg/min for 10 min IC), and L-NMMA+catalase+8-SPT. RESULTS: Cardiac tachypacing (60 to 120 beats/min) caused coronary vasodilation in both-sized arteries under control conditions in response to the increase in myocardial oxygen consumption. The metabolic coronary vasodilation was decreased after L-NMMA in subepicardial small arteries with an increased fluorescent H2O2 production compared with catalase group, whereas catalase decreased the vasodilation of arterioles with an increased fluorescent NO production compared with the L-NMMA group, and 8-SPT also decreased the vasodilation of arterioles. Furthermore, the metabolic coronary vasodilation was markedly attenuated after L-NMMA+catalase, L-NMMA+TEA, and L-NMMA+catalase+8-SPT in both-sized arteries. CONCLUSIONS: These results indicate that endogenous H2O2 plays an important role in pacing-induced metabolic coronary vasodilation in vivo.

Edaravone preserves coronary microvascular endothelial function after ischemia/reperfusion on the beating canine heart in vivo.

We examined whether edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, exerts its protective effect on coronary microvessels after ischemia/reperfusion (I/R) in vivo. Ninety-minute coronary occlusion followed by reperfusion was performed in 16 open-chest dogs with and without edaravone administration. Coronary small artery (> or = 100 microm in size) and arteriolar (< 100 microm) vasodilation, in the presence of endothelium-dependent (acetylcholine) or -independent (papaverine) vasodilators, was directly observed using intravital microscopy before and after I/R. I/R impaired microvascular vasodilation in response to acetylcholine, whereas administration of edaravone preserved the response in microvessels of both sizes, but to a greater extent in the coronary small arteries. No significant changes were noted with papaverine administration. In the edaravone group, the fluorescent intensity from reactive oxygen species (ROS) was lower, whereas nitric oxide (NO) intensity was higher relative to controls in the microvessels of the ischemic area. In conclusion, edaravone preserves coronary microvascular endothelial function after I/R in vivo. These effects, which were NO-mediated, were attributed to the ROS scavenging properties of edaravone.