Motoko Shiozaki

Morphological and biochemical signs of age-related neurodegenerative changes in klotho mutant mice

Klotho mutant mice, defective in the klotho gene, develop multiple age-related disorders with very short lifespans. Introduction of the exogenous klotho gene into these mutant mice leads to an improvement in their phenotypes, while overexpression of this gene in wild-type mice significantly extends their lifespan. These observations suggest that the klotho gene/protein has an anti-aging function. Since there have been only a few reports with some disagreement about results on the CNS of the mutant mice, we tried to clarify whether the CNS neurons generate aging-like features, even in premature stages, using biochemical and morphological approaches. Results obtained from the mutant mice, when compared with wild-type mice, were as follows. Neurofilaments (NFs) were increased significantly in axons, with the subunit proteins showing a significant enhancement in phosphorylation or expression of NF-H or NF-L, respectively. Microtubules in Purkinje cell dendrites were closer to each other, and in the CNS tissue tubulin was unaltered, but microtubule-associated protein (MAP) 2 was significantly reduced in expression. Neuronal cellular organelles were morphologically disordered. Lysosomes, cathepsin D and light chain 3 of MAP1A/B (LC3) were augmented with the appearance of putative autophagy-related structures. Antiapoptotic Bcl-xL and proapoptotic Bax were reduced and enhanced, respectively, and mitogen-activated protein kinase was reduced. Synapse-related proteins and structures were decreased. Neuronal degeneration was evident in hippocampal pyramidal cells, and possibly in Purkinje cells. Astrocytic glial filaments and glial fibrillary acidic protein were increased in density and expression, respectively. Together, the CNS neuronal alterations in klotho mutant mice were quite similar to those found in aged animals, including even premature death, so this mouse should be a more appropriate animal model for CNS aging than those previously reported.

Cell-sheet Therapy With Omentopexy Promotes Arteriogenesis and Improves Coronary Circulation Physiology in Failing Heart

Cell-sheet transplantation induces angiogenesis for chronic myocardial infarction (MI), though insufficient capillary maturation and paucity of arteriogenesis may limit its therapeutic effects. Omentum has been used clinically to promote revascularization and healing of ischemic tissues. We hypothesized that cell-sheet transplantation covered with an omentum-flap would effectively establish mature blood vessels and improve coronary microcirculation physiology, enhancing the therapeutic effects of cell-sheet therapy. Rats were divided into four groups after coronary ligation; skeletal myoblast cell-sheet plus omentum-flap (combined), cell-sheet only, omentum-flap only, and sham operation. At 4 weeks after the treatment, the combined group showed attenuated cardiac hypertrophy and fibrosis, and a greater amount of functionally (CD31(+)/lectin(+)) and structurally (CD31(+)/α-SMA(+)) mature blood vessels, along with myocardial upregulation of relevant genes. Synchrotron-based microangiography revealed that the combined procedure increased vascularization in resistance arterial vessels with better dilatory responses to endothelium-dependent agents. Serial (13)N-ammonia PET showed better global coronary flow reserve in the combined group, mainly attributed to improvement in the basal left ventricle. Consequently, the combined group had sustained improvements in cardiac function parameters and better functional capacity. Cell-sheet transplantation with an omentum-flap better promoted arteriogenesis and improved coronary microcirculation physiology in ischemic myocardium, leading to potent functional recovery in the failing heart.

Resveratrol affects undifferentiated and differentiated PC12 cells differently, particularly with respect to possible differences in mitochondrial and autophagic functions.

Since resveratrol is considered to exert a unique dual effect, protective for normal cells but toxic to tumor cells, its action on undifferentiated (original) and differentiated PC12 cells was analyzed, because undifferentiated cells are tumorigenic and differentiated ones are neuronal in nature. Compared to resveratrol-untreated cells in both undifferentiated and differentiated cell groups, cells treated with different doses of resveratrol, at dosages of 1, 10 and 100 μM, showed the following alterations. Dying/dead cells were significantly increased in a dose-dependent manner in undifferentiated cells, but they were unchanged at doses of up to 10 μM resveratrol in differentiated cells. In living cells, neurites were short in undifferentiated cells, but drastically elongated with an increased number in differentiated cells. The expression of SIRT1 was drastically reduced in undifferentiated cells, but stable in differentiated cells. SIRT3 was significantly enhanced in a dose-dependent manner at resveratrol doses of up to 10 μM in both cells, with reduction and more enhanced at a dosage of 100 μM in undifferentiated and differentiated cells, respectively. Mitochondrial number and ATP synthase β subunit expression was unaltered at doses of up to 10 μM and were significantly reduced at doses of 100 μM in undifferentiated cells, but they were significantly increased in a dose-dependent manner, with a slight reduction in the ATP synthase at doses of 100 μM, in differentiated cells. In a dose-dependent manner, the number of autophagosomes and the LC3-II/LC3-I ratio were significantly less in undifferentiated cells and greater in differentiated cells. Also, in a dose-dependent manner, the expression of phosphorylated AMP-activated kinase (AMPK) was significantly less in undifferentiated cells and greater in differentiated cells. Resveratrol-induced AMPK suppression and activation, possibly through the modulation of SIRT protein activity, may thus be related to the inhibition and promotion of mitochondrial and autophagic functions, leading to cell death and survival in undifferentiated and differentiated cells, respectively.

Very-high-dose α-tocopherol supplementation increases blood pressure and causes possible adverse central nervous system effects in stroke-prone spontaneously hypertensive rats.

Tocopherols and tocotrienols constitute the vitamin E family. Although α‐tocotrienol is the most neuroprotective form of vitamin E proved to be effective against stroke, α‐tocopherol is the most abundant in nature and is used most often for disease prevention/treatment. A recent metaanalysis of human studies suggested that α‐tocopherol supplementation increases all‐cause mortality. Therefore, we investigated the effects of α‐tocopherol (∼44 mg/kg body weight; equivalent to 2,600 mg/human/day) on the central nervous system (CNS) of stroke‐prone spontaneously hypertensive rats (SHRSP). SHRSP treated with high dose α‐tocopherol had significantly higher blood pressure than untreated controls fed a basal diet that contained ∼4 mg tocopherols/kg body weight, but neither group experienced a change in degree of lipid peroxidation in serum or CNS tissue. Biochemical/immunohistochemical analyses demonstrated that expressions of phosphorylated neurofilament H protein, glial fibrillary acidic protein and cathepsin D in the CNS tissue were significantly enhanced in α‐tocopherol‐supplemented rats, whereas expressions of SOD2 and Bcl‐xL were diminished in response to α‐tocopherol supplementation. Similarly, the frequency of cathepsin D‐positive cells, corresponding mostly to microglial cells, was significantly increased in α‐tocopherol‐supplemented rats. α‐Tocopherol supplementation also increased the number of lysosomes and lipofuscin granules in perikarya of both hippocampal pyramidal and Purkinje cells. Furthermore, α‐tocopherol supplementation increased the frequency of glial filaments and lipofuscin granules in astrocytes and lysosomes in microglial cells that were frequently occupied with phagocytosed inclusion structures. The present results are the first to suggest that a very high dose of α‐tocopherol supplementation increases blood pressure in SHRSP rats and influences the CNS tissue in a manner that seems adverse.

Hepatic gap junctions in the hepatocarcinogen-resistant DRH rat

Although the gap junction or connexin (Cx) is considered to be a tumor-suppressor, it is also required for tumor promotion. Therefore, we examined hepatic gap junctions in hepatocarcinogen-resistant (DRH) rats. Specifically, we investigated gap junction structure and Cx32 expression during normal conditions and in response to a hepatocarcinogen, 3′-methyl-4-dimethylaminoazobenzene (3′-MeDAB). On a basal diet without 3′-MeDAB, hepatic gap junctions and Cx32 protein expression were greater in DRH rats than in control Donryu rats, as evidenced by morphometry, immunohistochemistry and immunoblotting. On a diet containing 3′-MeDAB, gap junctions and expressed Cx32 were increased significantly in Donryu rats, but not in DRH rats. In this condition, Donryu rats lost weight but DRH rats increased relative liver weight. After 3′-MeDAB treatment, cathepsin D expression in hepatocytes was significantly increased only in Donryu rats, indicating that DRH rats were less susceptible to 3′-MeDAB. The abundance of mitogen-activated protein kinase, some constituent of which might be associated with the degree of Cx protein phosphorylation, was reduced to a greater extent in Donryu than in DRH rats after 3′-MeDAB treatment. The resistance of DRH rats to carcinogenesis may be due partially to their stabilized gap junctions, which could coordinate metabolic coupling to evade 3′-MeDAB toxicity.

A slow-releasing form of prostacyclin agonist (ONO1301SR) enhances endogenous secretion of multiple cardiotherapeutic cytokines and improves cardiac function in a rapid-pacing–induced model of canine heart failure

OBJECTIVES Cardiac functional deterioration in dilated cardiomyopathy (DCM) is known to be reversed by intramyocardial up-regulation of multiple cardioprotective factors, whereas a prostacyclin analog, ONO1301, has been shown to paracrinally activate interstitial cells to release a variety of protective factors. We here hypothesized that intramyocardial delivery of a slow-releasing form of ONO1301 (ONO1301SR) might activate regional myocardium to up-regulate cardiotherapeutic factors, leading to regional and global functional recovery in DCM. METHODS AND RESULTS ONO1301 elevated messenger RNA and protein level of hepatocyte growth factor, vascular endothelial growth factor, and stromal-derived factor-1 of normal human dermal fibroblasts in a dose-dependent manner in vitro. Intramyocardial delivery of ONO1301SR, which is ONO1301 mixed with polylactic and glycolic acid polymer (PLGA), but not that of PLGA only, yielded significant global functional recovery in a canine rapid pacing-induced DCM model, assessed by echocardiography and cardiac catheterization (n = 5 each). Importantly, speckle-tracking echocardiography unveiled significant regional functional recovery in the ONO1301-delivered territory, consistent to significantly increased vascular density, reduced interstitial collagen accumulation, attenuated myocyte hypertrophy, and reversed mitochondrial structure in the corresponding area. CONCLUSIONS Intramyocardial delivery of ONO1301SR, which is a PLGA-coated slow-releasing form of ONO1301, up-regulated multiple cardiotherapeutic factors in the injected territory, leading to region-specific reverse left ventricular remodeling and consequently a global functional recovery in a rapid-pacing-induced canine DCM model, warranting a further preclinical study to optimize this novel drug-delivery system to treat DCM.

Human Pluripotent Stem Cell-Derived Cardiac Tissue-like Constructs for Repairing the Infarcted Myocardium

Summary High-purity cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are promising for drug development and myocardial regeneration. However, most hiPSC-derived CMs morphologically and functionally resemble immature rather than adult CMs, which could hamper their application. Here, we obtained high-quality cardiac tissue-like constructs (CTLCs) by cultivating hiPSC-CMs on low-thickness aligned nanofibers made of biodegradable poly(D,L-lactic-co-glycolic acid) polymer. We show that multilayered and elongated CMs could be organized at high density along aligned nanofibers in a simple one-step seeding process, resulting in upregulated cardiac biomarkers and enhanced cardiac functions. When used for drug assessment, CTLCs were much more robust than the 2D conventional control. We also demonstrated the potential of CTLCs for modeling engraftments in vitro and treating myocardial infarction in vivo. Thus, we established a handy framework for cardiac tissue engineering, which holds high potential for pharmaceutical and clinical applications.

Histone Modification Is Correlated With Reverse Left Ventricular Remodeling in Nonischemic Dilated Cardiomyopathy

BACKGROUND Although implantation of a left ventricular assist device (LVAD) induces reverse remodeling of the left ventricle in end-stage nonischemic dilated cardiomyopathy (DCM), the underlying mechanism is not fully understood. It has been shown that epigenetic modification, such as methylation or acetylation of the histone, is one of the most important upstream signals in cardiac failure. This study hypothesized that histone profiles may be modified by LVAD implantation for end-stage nonischemic DCM, in association with reverse left ventricular remodeling. METHODS Hemodynamic changes associated with histone modification profiles in the left ventricle were comprehensively assessed in 14 patients with a diagnosis of end-stage nonischemic DCM. These patients underwent LVAD implantation and subsequent cardiac transplantation in our institution (Osaka University Hospital, Osaka, Japan). Samples of normal left ventricle from 3 different people were used as a control. RESULTS After LVAD support for 2.5 ± 1.2 years, the study cohort showed a significant reverse remodeling of left ventricular function associated with histopathologic changes in the left ventricle, such as reduction of myocyte size. Although the left ventricle of the cohort histologically expressed less 3 histone methylation-related molecules (eg, H3 lysine 4 trimethylation [H3K4me3], H3 lysine 9 dimethylation [H3K9me2], and H3 lysine 9 trimethylation [H3K9me3]) compared with normal left ventricle, LVAD support reversed expression of these molecules, associated with up-regulation of H3 lysine 9 [H3K9] methyltransferase and suppressor of variegation 3-9 homologue 1 [SUV39H1] and with down-regulation of H3K9 demethylase and jumonji domains [JMJDs] in the LVAD-supported left ventricle. Moreover, expression of atrial natriuretic peptide and brain natriuretic peptide (ANP and BNP) was negatively correlated with that of H3K9me2 and H3K9me3. CONCLUSIONS The epigenetic state of cardiac myocytes (eg, as histone methylation) was substantially modulated in end-stage nonischemic DCM. LVAD support partially reversed the epigenetic state and its upstream signals, in association with pathologic and functional reverse remodeling.

Cordyceps militaris improves the survival of Dahl salt-sensitive hypertensive rats possibly via influences of mitochondria and autophagy functions

The genus Cordyceps and its specific ingredient, cordycepin, have attracted much attention for multiple health benefits and expectations for lifespan extension. We analyzed whether Cordyceps militaris (CM), which contains large amounts of cordycepin, can extend the survival of Dahl salt-sensitive rats, whose survival was reduced to ∼3 months via a high-salt diet. The survival of these life-shortened rats was extended significantly when supplemented with CM, possibly due to a minimization of the effects of stroke. Next, we analyzed the effect of CM on hypertension-sensitive organs, the central nervous systems (CNS), heart, kidney and liver of these rats. We attempted to ascertain how the organs were improved by CM, and we paid particular attention to mitochondria and autophagy functions. The following results were from CM-treated rats in comparison with control rats. Microscopically, CNS neurons, cardiomyocytes, glomerular podocytes, renal epithelial cells, and hepatocytes all were improved. However, immunoblot and immunohistochemical analysis showed that the expressions of mitochondria-related proteins, ATP synthase β subunit, SIRT3 and SOD2, and autophagy-related proteins, LC3-II/LC3-I ratio and cathepsin D all were reduced significantly in the CNS neurons, but increased significantly in the cells of the other three organs, although p62 was decreased in its expression in all the organs tested. Activity of Akt and mTOR was enhanced but that of AMPK was reduced in the CNS, while such kinase activity was completely the opposite in the other organs. Together, the influence of CM may differ between mitochondria and autophagy functioned between the two organ groups, as mitochondria and autophagy seemed to be repressed and promoted, respectively, in the CNS, while both mitochondria and autophagy were activated in the others. This could possibly be related to the steady or improved cellular activity in both the organs, which might result in the life extension of these rats.

Protective effects of xanthosine on ethanol-impaired neurons

Xanthosine, a component of roasted rice bran extracts, is not polyphenol but has a potent antioxidant activity. We analyzed whether this chemical alleviates neuronal damage induced by a high dosage of ethanol. Rats were treated with 20% ethanol plus 0.1% xantosine for 42 weeks from the age of 6 weeks, and their CNS neurons were compared with those treated with or without 20% ethanol alone by immunoblotting, immunohistochemistry and electron microscopy. Addition of xanthosine to ethanol reduced distribution density of hepatic lipid droplets augmented by ethanol. In the CNS, xanthosine reduced appearance of lipofuscin granules and neurofilaments, markers for the neuronal damage, enhanced expressions of synaptophysin, superoxide dismutase and Bcl-xL related with activation of neuronal function, and decreased neurofilament proteins, GFAP and Bax associated with neurodeterioration. We first demonstrate that xanthosine is beneficial to the neurons.