Hirotaka Iwasaki

Different influences of extracellular and intracellular superoxide on relaxation through the NO/sGC/cGMP pathway in isolated rat iliac arteries.

Abstract: Superoxide production is increased in diseased blood vessels, which is considered to lead to impairment of the nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP pathway. To investigate the respective influence of extracellular and intracellular superoxide on vascular function through the NO/sGC/cGMP pathway, mechanical responses of rat external iliac arteries without endothelium were studied under exposure to a superoxide-generating agent, pyrogallol, or menadione. Exposure to pyrogallol impaired the relaxation induced by acidified NaNO2 (exogenous NO) but not that by nitroglycerin (organic nitrate), BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator), or 8-Br-cGMP (cGMP analog). Superoxide dismutase (SOD) and tempol restored the impaired relaxation by acidified NaNO2. Superoxide production in the bathing solution, but not in artery segments, was significantly increased by exposure to pyrogallol, which was abolished in the presence of SOD or tempol. However, exposure to menadione impaired the relaxant response to acidified NaNO2, nitroglycerin, or BAY 41-2272, whereas it augmented that to BAY 60-2770. Also, this exposure had no effect on the 8-Br-cGMP-induced vasorelxation. Superoxide production in artery segments was dramatically enhanced by exposure to menadione, whereas that in the bathing solution was not affected. This increase in vascular superoxide production was normalized by tempol but not by SOD. These findings suggest that extracellular superoxide reacts with NO only outside the cell, whereas intracellular superoxide not only scavenges NO inside the cell but also shifts the sGC redox equilibrium.

Effects of Peroxynitrite on Relaxation through the NO/sGC/cGMP Pathway in Isolated Rat Iliac Arteries

Background/Aims: The present study investigated the mechanism by which peroxynitrite impairs vascular function through the nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP pathway. Methods: Mechanical responses of rat external iliac arteries without endothelium were studied under exposure to peroxynitrite. cGMP concentrations were determined by enzyme immunoassay. Results: Relaxation induced by BAY 41-2272 (sGC stimulator) was impaired under exposure to peroxynitrite, whereas that by BAY 60-2770 (sGC activator) was enhanced. These responses were correlated with tissue levels of cGMP. Effects of peroxynitrite on the relaxant responses to BAY compounds were also observed in the presence of superoxide dismutase (SOD) or tempol, both of which scavenge a certain kind of reactive molecules other than peroxynitrite. As is the case with the relaxant response to BAY 41-2272, acidified NaNO2- and nitroglycerin-induced relaxations were markedly attenuated by exposing the arteries to peroxynitrite, which was not abolished by preincubation with SOD or tempol. On the other hand, peroxynitrite exposure had no effect on the 8-Br-cGMP-induced vasorelxation. Conclusion: These findings suggest that peroxynitrite interferes with the NO/sGC/cGMP pathway by altering the redox state of sGC. It is likely that peroxynitrite can shift the sGC redox equilibrium to the NO-insensitive state in the vasculature.

Effects of hydrogen peroxide on relaxation through the NO/sGC/cGMP pathway in isolated rat iliac arteries

Abstract The production of reactive oxygen species, including hydrogen peroxide (H2O2), is increased in diseased blood vessels. Although H2O2 leads to impairment of the nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP signaling pathway, it is not clear whether this reactive molecule affects the redox state of sGC, a key determinant of NO bioavailability. To clarify this issue, mechanical responses of endothelium-denuded rat external iliac arteries to BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator), nitroglycerin (NO donor), acidified NaNO2 (exogenous NO) and 8-Br-cGMP (cGMP analog) were studied under exposure to H2O2. The relaxant response to BAY 41-2272 (pD2: 6.79 ± 0.10 and 6.62 ± 0.17), BAY 60-2770 (pD2: 9.57 ± 0.06 and 9.34 ± 0.15) or 8-Br-cGMP (pD2: 5.19 ± 0.06 and 5.24 ± 0.08) was not apparently affected by exposure to H2O2. In addition, vascular cGMP production stimulated with BAY 41-2272 or BAY 60-2770 in the presence of H2O2 was identical to that in its absence. On the other hand, nitroglycerin-induced relaxation was markedly attenuated by exposing the arteries to H2O2 (pD2: 8.73 ± 0.05 and 8.30 ± 0.05), which was normalized in the presence of catalase (pD2: 8.59 ± 0.05). Likewise, H2O2 exposure impaired the relaxant response to acidified NaNO2 (pD2: 6.52 ± 0.17 and 6.09 ± 0.16). These findings suggest that H2O2 interferes with the NO-mediated action, but the sGC redox equilibrium and the downstream target(s) of cGMP are unlikely to be affected in the vasculature.

Duality of n-3 Polyunsaturated Fatty Acids on Mcp-1 Expression in Vascular Smooth Muscle: A Potential Role of 4-Hydroxy Hexenal

N-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have protective effects against atherosclerosis. Monocyte chemotactic protein (MCP)-1 is a major inflammatory mediator in the progression of atherosclerosis. However, little is known about the regulation of Mcp-1 by DHA and EPA in vessels and vascular smooth muscle cells (VSMCs). In this study, we compared the effect of DHA and EPA on the expression of Mcp-1 in rat arterial strips and rat VSMCs. DHA, but not EPA, suppressed Mcp-1 expression in arterial strips. Furthermore, DHA generated 4-hydroxy hexenal (4-HHE), an end product of n-3 polyunsaturated fatty acids (PUFAs), in arterial strips as measured by liquid chromatography-tandem mass spectrometry. In addition, 4-HHE treatment suppressed Mcp-1 expression in arterial strips, suggesting 4-HHE derived from DHA may be involved in the mechanism of this phenomenon. In contrast, Mcp-1 expression was stimulated by DHA, EPA and 4-HHE through p38 kinase and the Keap1-Nuclear factor erythroid-derived 2-like 2 (Nrf2) pathway in VSMCs. In conclusion, there is a dual effect of n-3 PUFAs on the regulation of Mcp-1 expression. Further study is necessary to elucidate the pathological role of this phenomenon.

MicroRNA-494 plays a role in fiber type-specific skeletal myogenesis in human induced pluripotent stem cells

Mitochondrial oxidative capacity in skeletal muscle is known to decrease in diabetic patients, and sarcopenia is a risk factor for diabetes, particularly in elderly people. We previously revealed that microRNA (miR)-494 inhibits mitochondrial biogenesis during myogenic differentiation in murine C2C12 cells and others reported that exercise regulates miR-494 levels in obese sedentary individuals with increased risk of type 2 diabetes. In this study, to investigate the therapeutic potential of miR-494, we first investigated the role of miR-494 during human skeletal myogenesis. Using human induced pluripotent stem (hiPS) cells stably transfected with the Tet/ON-myogenic differentiation 1(MYOD1) gene (MyoD-hiPS cells), we found that miR-494 expression transiently increased and was downregulated after myogenic induction. In miR-494 transfected MyoD-hiPS cells, the level of high oxidative fiber (type IIa) marker proteins specifically decreased, while no change in the total number of cells was observed. In contrast, the expression of both type I and type IIx markers was unaffected by miR-494 overexpression. Furthermore, miR-494 overexpression suppressed basal oxygen consumption rate concomitant with the inhibition of myotube formation and without significant effects on the mitochondrial content. These results suggest that miR-494 plays a novel role in the fiber type-specific skeletal myogenesis in MyoD-hiPS cells, distinct from murine C2C12 myogenesis.

Aging does not affect soluble guanylate cyclase redox state in mouse aortas

Aging is associated with endothelial dysfunction, defined as a reduction in nitric oxide (NO) bioavailability. Although the redox state of the NO acceptor soluble guanylate cyclase (sGC) is another determinant factor for its bioavailability and is disturbed by reactive oxygen species (ROS) known to be increased with age, it is unclear whether aging actually has an impact on vascular sGC redox equilibrium. Therefore, this study investigated this issue using two different types of compounds, the sGC stimulator BAY 41‐2272 and the sGC activator BAY 60‐2770. Plasma thiobarbituric acid‐reactive substances (TBARS) levels were markedly higher in aged (19–20 months old) mice than in young (2–3 months old) mice, whereas superoxide levels in endothelium‐denuded aortas were not different between the groups. The relaxant response of endothelium‐denuded aortas to either BAY 41‐2272 or BAY 60‐2770 was identical in aged and young mice. In addition, the vascular cGMP production stimulated with BAY 41‐2272 or BAY 60‐2770 in aged mice was the same level as that in young mice. These findings suggest that aging accompanied by an increase in systemic oxidative stress does not affect vascular smooth muscle ROS generation and sGC redox equilibrium. Unless ROS are increased in vascular smooth muscle, the sGC redox equilibrium might remain unchanged.

MiR-494-3p regulates mitochondrial biogenesis and thermogenesis through PGC1-α signalling in beige adipocytes

Mitochondria are critical in heat generation in brown and beige adipocytes. Mitochondrial number and function are regulated in response to external stimuli, such as cold exposure and β3 adrenergic receptor agonist. However, the molecular mechanisms regulating mitochondrial biogenesis during browning, especially by microRNAs, remain unknown. We investigated the role of miR-494-3p in mitochondrial biogenesis during adipogenesis and browning. Intermittent mild cold exposure of mice induced PPARγ coactivator1-α (PGC1-α) and mitochondrial TFAM, PDH, and ANT1/2 expression along with uncoupling protein-1 (Ucp1) in inguinal white adipose tissue (iWAT). miR-494-3p levels were significantly downregulated in iWAT upon cold exposure (p < 0.05). miR-494-3p overexpression substantially reduced PGC1-α expression and its downstream targets TFAM, PDH and MTCO1 in 3T3-L1 white and beige adipocytes (p < 0.05). miR-494-3p inhibition in 3T3-L1 white adipocytes resulted in increased PDH (p < 0.05). PGC1-α, TFAM and Ucp1 mRNA levels were robustly downregulated by miR-494-3p overexpression in 3T3-L1 beige adipocytes, along with strongly decreased oxygen consumption rate. PGC1-α and Ucp1 proteins were downregulated by miR-494-3p in primary beige cells (p < 0.05). Luciferase assays confirmed PGC1-α as a direct gene target of miR-494-3p. Our findings demonstrate that decreased miR-494-3p expression during browning regulates mitochondrial biogenesis and thermogenesis through PGC1-α.