Hirofumi Machii

Aging impairs myocardium-induced dilation in coronary arterioles: Role of hydrogen peroxide and angiotensin

The mechanisms that drive age-related modifications of coronary circulation by myocardium have not been fully defined. To elucidate the aging effect on myocyte-induced vascular response, we measured changes in the diameter of isolated coronary arterioles to supernatant collected from isolated cardiac myocytes of young (2 mo) and old (24 mo) rats (stimulated at 400 beats/min, n=10, each). The H(2)O(2) level in pacing myocyte supernatant was greater in old rats than in young ones (15.9±1.8 vs. 9.5±0.7μM, P<0.01). Catalase activity in myocytes decreased 38.6±5.2% in old rats compared to that in young rats. Vasodilation with young-myocyte supernatant (M) (response to 500μl; young-arterioles (A) 20.5±1.6%, old-A 18.2±1.2%) was more potent compared to that with old-M (young-A 10.3±0.8%, old-A 9.4±1.0%, P<0.01, respectively). Treatment with an angiotensin II receptor antagonist, olmesartan, in a vessel bath augmented vasodilation in old-M+young-A (34.9±4.0%, P<0.01) and old-M+old-A (27.2±2.8%, P<0.01). Administration of catalase converted vasodilation to vasoconstriction in old-M and eliminated vasodilation in young-M. Vascular responses with authentic H(2)O(2) and angiotensin II were similar between old- and young-A. Thus, aging increases both angiotensin and the H(2)O(2) release from myocardium. In conclusion, cardiac myocyte-dependent signaling plays an important role in determining coronary vascular tone in the aging heart.

Epicardial Adipose Tissue Reflects the Presence of Coronary Artery Disease: Comparison with Abdominal Visceral Adipose Tissue

Accumulation of visceral adipose tissue is associated with a risk of coronary artery disease (CAD). The aim of this study was to examine whether different types of adipose tissue depot may play differential roles in the progression of CAD. Consecutive 174 patients who underwent both computed tomography (CT) and echocardiography were analyzed. Cardiac and abdominal CT scans were performed to measure epicardial and abdominal visceral adipose tissue (EAT and abdominal VAT, resp.). Out of 174 patients, 109 and 113 patients, respectively, presented coronary calcification (CC) and coronary atheromatous plaque (CP). The EAT and abdominal VAT areas were larger in patients with CP compared to those without it. Interestingly, the EAT area was larger in patients with CC compared to those without CC, whereas no difference was observed in the abdominal VAT area between patients with CC and those without. Multivariable logistic regression analysis revealed that the presence of echocardiographic EAT was an independent predictor of CP and CC, but the abdominal VAT area was not. These results suggest that EAT and abdominal VAT may play differential pathological roles in CAD. Given the importance of CC and CP, we should consider the precise assessment of CAD when echocardiographic EAT is detected.

Coronary Artery Spasm Related to Thiol Oxidation and Senescence Marker Protein-30 in Aging

BACKGROUND Senescence marker protein-30 (SMP30) decreases with aging, and SMP30 knockout (KO) mice show a short life with increased oxidant stress. AIMS We assessed the effect of oxidant stress with SMP30 deficiency in coronary artery spasm and clarify its underlying mechanisms. RESULTS We measured vascular responses to acetylcholine (ACh) and sodium nitroprusside (SNP) of isolated coronary arteries from SMP30 KO and wild-type (WT) mice. In SMP30 KO mice, ACh-induced vasoconstriction occurred, which was changed to vasodilation by dithiothreitol (DTT), a thiol-reducing agent. However, Nω-nitro-L-arginine-methyl ester, nitric oxide (NO) synthase inhibitor, or tetrahydrobiopterin did not change the ACh response. In isolated coronary arteries of WT mice, ACh-induced vasodilation occurred. Inhibition of glutathione reductase by 1, 3-bis(2-chloroethyl)-1-nitrosourea decreased ACh-induced vasodilation (n=10, p<0.01), which was restored by DTT. To evaluate the thiol oxidation, we measured the fluorescence of monochlorobimane (MCB) in coronary arteries, which covalently labels the total. The fluorescence level to MCB decreased in SMP30 KO mice, but with DTT treatment restored to a level comparable to that of WT mice. The reduced glutathione and total thiol levels were also low in the aorta of SMP30 KO mice compared with those of WT mice. Administration of ACh into the aortic sinus in vivo of SMP30 KO mice induced coronary artery spasm. INNOVATION The thiol redox state is a key regulator of endothelial NO synthase activity, and thiol oxidation was associated with endothelial dysfunction in the SMP30 deficiency model. CONCLUSION These results suggest that chronic thiol oxidation by oxidant stress is a trigger of coronary artery spasm, resulting in impaired endothelium-dependent vasodilation.

Metabolic regulation of coronary vascular tone: role of hydrogen peroxide, purinergic components, and angiotensin.

Metabolic regulation plays an important role in modifying coronary vascular tone. We hypothesized that hydrogen peroxide, purinergic components, and angiotensin, produced by cardiac myocytes control coronary vascular tone in proportion to metabolism. We measured changes in the diameter of isolated, pressurized coronary arterioles in response to supernatant from isolated cardiac myocytes in rats (stimulated for 20-, 60-, and 120-min at 400 bpm). Changes in the diameter of arterioles were determined under control conditions following treatment of arterioles with an adenosine receptor antagonist, 8-PSPT, a P2Y1 receptor antagonist, MRS-2179, or an angiotensin II receptor antagonist, olmesartan. A supernatant (500 microl to a 2 ml bath) from myocytes stimulated for 20-, 60- and 120-min caused graded vasodilation (14.1+/-0.4, 20.2+/-1.6, 53.8+/-6.2%, P<0.01 vs. non-stimulated, respectively). In 20-min stimulation, catalase with myocyte supernatants eliminated vasodilation. Following 60-min stimulation, catalase converted myocyte supernatant-induced vasodilation to a vasoconstriction (-15.1+/-1.0%), and this vasoconstriction was eliminated by olmesartan. Upon 120-min stimulation, catalase partially reduced the vasodilation by myocyte supernatants (37.2+/-3.8%). The remaining vasodilation was converted to a vasoconstriction with 8-PSPT and MRS-2179, and this vasoconstriction was completely eliminated with olmesartan. Cardiac myocytes modulate vascular tone through the net effects of hydrogen peroxide, purinergic components (adenosine and ADP), and angiotensin in proportion to ischemia.

Mitochondrial-Targeted Antioxidant Maintains Blood Flow, Mitochondrial Function, and Redox Balance in Old Mice Following Prolonged Limb Ischemia

Aging is a major factor in the decline of limb blood flow with ischemia. However, the underlying mechanism remains unclear. We investigated the role of mitochondrial reactive oxygen species (ROS) with regard to limb perfusion recovery in aging during ischemia. We performed femoral artery ligation in young and old mice with or without treatment with a scavenger of mitochondrial superoxide, MitoTEMPO (180 μg/kg/day, from pre-operative day 7 to post-operative day (POD) 21) infusion using an implanted mini-pump. The recoveries of cutaneous blood flow in the ischemic hind limb were lower in old mice than in young mice but were improved in MitoTEMPO-treated old mice. Mitochondrial DNA damage appeared in ischemic aged muscles but was eliminated by MitoTEMPO treatment. For POD 2, MitoTEMPO treatment suppressed the expression of p53 and the ratio of Bax/Bcl2 and upregulated the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in ischemic aged skeletal muscles. For POD 21, MitoTEMPO treatment preserved the expression of PGC-1α in ischemic aged skeletal muscle. The ischemic soleus of old mice showed a lower mitochondrial respiratory control ratio in POD 21 compared to young mice, which was recovered in MitoTEMPO-treated old mice. Scavenging of mitochondrial superoxide attenuated mitochondrial DNA damage and preserved the mitochondrial respiration, in addition to suppression of the expression of p53 and preservation of the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) in ischemic skeletal muscles with aging. Resolution of excessive mitochondrial superoxide could be an effective therapy to recover blood flow of skeletal muscle during ischemia in senescence.

Resolution of mitochondrial oxidant stress improves aged-cardiovascular performance.

Background Senescence is a major factor that increases oxidative stress in mitochondria, which contributes toward the pathogenesis of heart disease. However, the effect of antioxidant therapy on cardiac mitochondria in aged-cardiac performance remains elusive. Objectives We postulated that the mitochondrial targeting of superoxide scavenging would have benefits in the aged heart. Methods and results Generation of superoxide in the mitochondria and nicotinamide adenine dinucleotide phosphate oxidase activity increased in the heart of old mice compared with that in young mice. In old mice treated with a mitochondria-targeted antioxidant MitoTEMPO (180 µg/kg/day, 28 days) co-infusion using a subcutaneously implanted minipump, levels of superoxide in the mitochondria and nicotinamide adenine dinucleotide phosphate oxidase activity as well as hydrogen peroxide decreased markedly in cardiomyocytes. Treatment with MitoTEMPO in old mice improved the systolic and diastolic function assessed by echocardiography. Endothelium-dependent vasodilation in isolated coronary arteries and endothelial nitric-oxide synthase phosphorylation were impaired in old mice compared with that in young mice and were improved by MitoTEMPO treatment. Mitochondria from the old mice myocardium showed lower rates of complex I-dependent and II-dependent respiration compared with that from young mice. Supplementation of MitoTEMPO in old mice improved the respiration rates and efficiency of ATP generation in mitochondria to a level similar to that of young mice. Conclusion Resolution of oxidative stress in mitochondria by MitoTEMPO in old mice restored cardiac function and the capacity of coronary vasodilation to the same magnitude observed in young mice. An antioxidant strategy targeting mitochondria could have a therapeutic benefit in heart disease with senescence.

Senescence marker protein-30 deficiency impairs angiogenesis under ischemia

Aging decreases collateral-dependent flow recovery following acute arterial obstruction. However, the mechanisms are partially understood, therefore critical management has been lacked in clinical setting. Senescence marker protein-30 (SMP30) is a novel aging marker, which is assumed to act as an anti-aging factor in various organs. Therefore, we studied the effect of SMP30 on ischemia-induced collateral growth in SMP30 knockout (KO) mice, young and old C57BL/6 mice. The SMP30 expression in gastrocnemius tissue was decreased in old mice compared to that of young mice. The recovery of cutaneous blood flow in hind limb after femoral artery ligation and tissue capillary density recoveries were suppressed in SMP30 KO and old mice compared to those in young mice. Nitric oxide generation induced by l-arginine and GSH/GSSG in aorta of SMP30 KO and old mice were lower than those in young mice. The levels of NADPH oxidase activity and superoxide production in the ischemic tissue were higher in SMP30 KO and old mice than in young mice. The phosphorylated eNOS and Akt levels and VEGF levels in ischemic muscle were lower in SMP30 KO and old mice than in young mice. Deficiency of SMP30 exacerbates oxidative stress related to NADPH oxidase activity enhancement and impairs eNOS activity, which leads to rarefaction of angiogenesis induced by ischemia. These results suggest that SMP30 plays a key role in disrupting collateral growth under ischemia in aging.

Age-related oxidant stress with senescence marker protein-30 deficiency plays a pivotal role in coronary artery spasm.

ObjectivesWe examined the mechanism of coronary artery spasm related to oxidant stress with aging in senescence marker protein-30 (SMP30)-deficient mice because SMP30 decreases with aging and SMP30 knockout (KO) mice show a short life with increased oxidant stress. MethodsTo examine the effect of SMP30 on coronary artery vasomotor tone, we measured the endothelium-dependent [5-hydroxytryptamine (5-HT)] response of isolated, pressurized coronary arteries from SMP30 KO and wild-type (WT) mice (n=10 each). ResultsIn SMP30 KO mice, 5-HT-induced vasoconstriction occurred, which altered vasodilation with dithiothreitol, a thiol-reducing agent. In WT mice, 5-HT-induced vasodilation occurred. Administration of 5-HT from the aortic sinus induced a coronary artery spasm in SMP30 KO mice, which was prevented by the intravenous administration of Y-27632, rho-kinase inhibitor. The fluorescence level of monochlorobimane in coronary arteries, which covalently labels the reduced total thiols, decreased in SMP30 KO mice, but reverted to a level comparable with that of WT mice on treatment with Y-27632. From these results, SMP30 provides protection against coronary artery spasm. ConclusionChronic oxidant stress associated with aging plays an important role in coronary artery spasm related to thiol oxidation and rho-kinase signaling.

Predominance of Abdominal Visceral Adipose Tissue Reflects the Presence of Aortic Valve Calcification

Background. Aortic valve calcification (AVC) is a common feature of aging and is related to coronary artery disease. Although abdominal visceral adipose tissue (VAT) plays fundamental roles in coronary artery disease, the relationship between abdominal VAT and AVC is not fully understood. Methods. We investigated 259 patients who underwent cardiac and abdominal computed tomography (CT). AVC was defined as calcified lesion on the aortic valve by CT. %abdominal VAT was calculated as abdominal VAT area/total adipose tissue area. Results. AVC was detected in 75 patients, and these patients showed higher %abdominal VAT (44% versus 38%, p < 0.05) compared to those without AVC. When the cutoff value of %abdominal VAT was set at 40.9%, the area under the curve to diagnose AVC was 0.626. Multivariable logistic regression analysis showed that age (OR 1.120, 95% CI 1.078–1.168, p < 0.01), diabetes (OR 2.587, 95% CI 1.323–5.130, p < 0.01), and %abdominal VAT (OR 1.032, 95% CI 1.003–1.065, p < 0.05) were independent risk factors for AVC. The net reclassification improvement value for detecting AVC was increased when %abdominal VAT was added to the model: 0.5093 (95% CI 0.2489–0.7697, p < 0.01). Conclusion. We determined that predominance of VAT is associated with AVC.

Giant left atrial thrombus caused by direct invasion of lung cancer

A 60-year-old man came to our hospital with dyspnea and was found at the front of our hospital due to syncope. He had cystic lung disease and a 2-month history of progressively worsening dyspnea. He regained consciousness in the emergency room; however, his lips were cyanotic and oxygen saturation was 70 %. He was afebrile, with a blood pressure of 116/79 mmHg and a heart rate of 70 bpm. There was no heart murmur, moist rale, or peripheral edema. Chest radiography showed pulmonary edema with bilateral pleural effusion. Electrocardiogram showed biphasic pulmonary P waves with regular sinus rhythm. Echocardiography demonstrated that the left atrium (LA) was occupied by a giant mass (7.6 9 4.8 cm, Fig. 1a, b). The mass was of low density and homogenous, and the mobility was poor. It was unclear whether the mass had a stalk or was attached on the LA wall. Color Doppler method revealed that the mass disturbed the left ventricular inflow (Fig. 1c, d). The mass was presumed to be a myxoma because he had been asymptomatic with sinus rhythm for a long time. He was referred to another hospital for emergency surgery. Just after the transfer, he was examined by chest enhanced computed tomography (CT) in the emergency room. The findings indicated a right lung cancer (Fig. 2, yellow arrow). The tumor directly infiltrated the wall of the LA. In addition, the mass in the LA, which was of homogenous low density without enhancement, was suspected to be thrombus (Fig. 2, white arrow). By the findings of a blood sampling examination (FDP 6.4 lg/ml, D-dimer 4.0 lg/ml), it was suspected that there was intracardiac thrombus, chronic DIC state, and/or deep vein thrombosis. He was diagnosed as having primary lung cancer with direct invasion into the LA and thrombus of the LA. Emergency surgery was considered; however, he was treated conservatively because the lung cancer was of stage IV. He was kept under sedation due to worsening dyspnea and eventually died of respiratory and circulatory disturbance on the 6th day of hospitalization. The pathological findings were: (1) primary lung adenocarcinoma in the right hilum pulmonis and the lower lobe with direct invasion to the LA and (2) stratified thrombus in the LA (the core of thrombus was the carcinoma cells in the LA). This is a rare case of a giant LA thrombus with direct invasion of lung cancer into the left atrium. The thrombus was first suspected as myxoma because of the patient’s history of illness and the echocardiographic findings (low density, homogenous) without atrial fibrillation. It seems that projecting carcinoma cells to the LA became the core and the LA thrombus stratified as it grows into a chronic DIC state based on malignancy. Few cases of intracardiac extension of lung cancer [1] and LA thrombus caused by direct invasion [2] have been reported. In urgent patients with circulatory disturbance, we have to make a decision M. Iwai-Takano (&) Intensive Care Unit, Fukushima Medical University Hospital, 1 Hikarigaoka, Fukushima 960-1295, Japan e-mail: masumi@fmu.ac.jp