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Biomedical subjects

R Nagai

Publications and source records attributed to R Nagai.

At least 19 recordsLinked to original sources
PubMed2000

Serum level of vascular endothelial growth factor is decreased by hormone replacement therapy in postmenopausal women without hypercholesterolemia.

The administration of hormone replacement therapy (HRT) to postmenopausal women (PMW) reportedly has beneficial effects on their levels of lipid and lipoproteins. Estrogen retards the development of atherosclerosis induced by a high-fat diet in animals. Although vascular endothelial growth factor (VEGF) may be involved in the development of atherosclerosis in humans, there is no information on effect of estrogen administration on VEGF level and lipid metabolism. We evaluated 64 healthy normotensive or hypertensive PMW before and during the administration of HRT (0.625 mg conjugated equine estrogen combined with 2.5 mg medroxyprogesterone acetate orally) daily for 6 months. All hypertensive PMW were well-controlled on antihypertensive drug therapy. According to their total cholesterol level at baseline, we divided the PMW with HRT (n=54) into a normocholesterolemic group (NC, total cholesterol <220 mg/dl, n=35) and a hypercholesterolemic group (HC, total cholesterol >/=220 mg/dl, n=19). We evaluated the serum levels of VEGF at baseline, and again at 3 and 6 months after starting HRT. HRT significantly (P<0.01) reduced the mean VEGF level from 31.5+/-4.3 pg/ml at baseline to 18.2+/-2.3 pg/ml after 6 months in the NC group. However, the VEGF levels in the HC group and the control group exhibited no significant change at either 3 or 6 months after starting HRT. In summary, HRT, using a combination of conjugated equine estrogen and medroxyprogesterone acetate, reduced the level of VEGF in normocholesterolemic PMW more effectively than in those with hypercholesterolemia.

Aged↗
PubMed1999

Sphingosylphosphorylcholine induces a hypertrophic growth response through the mitogen-activated protein kinase signaling cascade in rat neonatal cardiac myocytes.

The sphingolipid metabolites, sphingosine (SPH), SPH 1-phosphate (S1P), and sphingosylphosphorylcholine (SPC), can act as intracellular as well as extracellular signaling molecules. These compounds have been implicated in the regulation of cell growth, differentiation, and programmed cell death in nonmyocytes, but the effects of sphingolipid metabolites in cardiac myocytes are not known. Cultured neonatal rat cardiac myocytes were stimulated with SPH (1 to 10 micromol/L), S1P (1 to 10 micromol/L), or SPC (0.1 to 10 micromol/L) for 24 hours to determine the effects of sphingolipid metabolites on the rates of protein synthesis and degradation. Stimulation with SPC led to an increase in the total amount of protein, an accelerated rate of total protein synthesis, and a decrease in protein degradation in a dose-dependent manner. However, S1P had little effect and SPH had no effect on total protein synthesis. In addition, stimulation with SPC led to a 1.4-fold increase in myocardial cell size and enhanced atrial natriuretic factor gene expression. Pretreatment of the cardiac myocytes with pertussis toxin or PD98059 attenuated the SPC-induced hypertrophic growth response. Further, stimulation with SPC increased phosphorylation of mitogen-activated protein kinase (MAPK) and stimulated MAPK enzyme activity. Finally, endothelin-1 stimulated the generation of SPC in cardiac myocytes. The observation that SPC induces a hypertrophic growth response in cardiac myocytes suggests that SPC may play a critical role in the development of cardiac hypertrophy. The effects of SPC could be mediated, in part, by activation of a G protein-coupled receptor and a MAPK signaling cascade.

Adrenergic beta-Agonists↗
PubMed1999

MAPK superfamily plays an important role in daunomycin-induced apoptosis of cardiac myocytes.

BACKGROUND: Although anthracyclines, such as daunomycin (DM) and adriamycin, are potent chemotherapeutic agents, they have serious adverse effects, including cardiac toxicity. In the present study, we investigated the molecular mechanisms of DM-induced cardiomyocyte impairment. METHODS AND RESULTS: When cultured cardiac myocytes of neonatal rats were exposed to 1 micromol/L DM for 24 hours, many cells became positive for TUNEL staining, with morphological changes characteristic of apoptosis. Fragmentation of DNA into oligonucleosome-size fragments was recognized by agarose gel electrophoresis in DM-treated myocytes. DM activated 3 members of the mitogen-activated protein kinase (MAPK) family dose-dependently, such as extracellular signal-regulated protein kinases (ERKs), c-Jun NH(2)-terminal kinases, and p38 MAPK in cardiac myocytes. Oxyradical scavengers or Ca(2+) chelators inhibited DM-induced activation of ERKs and p38 MAPK. DM-induced activation of ERKs was also inhibited by overexpression of dominant negative mutants of Ras (D.N.Ras), and the p38 MAPK activation was attenuated by D.N.Rho. The number of DM-induced apoptotic cells was markedly increased when the ERK signaling pathway was selectively blocked by a specific MAPK/ERK kinase inhibitor, PD98059, whereas pretreatment with a specific inhibitor of p38 MAPK, SB203580, significantly reduced the amount of apoptosis. CONCLUSIONS: These results suggest that DM activates MAPKs through reactive oxygen species and Ca(2+) and that the MAPK family plays important roles in DM-induced apoptosis in cardiac myocytes. ERKs protect cardiomyocytes from apoptosis, whereas p38 MAPK is involved in the induction of cardiomyocyte apoptosis.

Animals↗
PubMed1999

Transcriptional activation of the zinc finger transcription factor BTEB2 gene by Egr-1 through mitogen-activated protein kinase pathways in vascular smooth muscle cells.

We have recently demonstrated that a developmentally regulated zinc finger protein, basic transcription regulatory element binding protein 2 (BTEB2), is induced in neointimal smooth muscle in response to vascular injury. In this study, we investigated the molecular mechanisms regulating BTEB2 expression in vascular smooth muscle cells (SMCs) in vitro. BTEB2 mRNA expression is rapidly and persistently induced in SMCs by phorbol 12-myristate 13-acetate (PMA) and basic fibroblast growth factor. We have isolated and characterized the promoter region of the human BTEB2 gene to determine the regulatory network controlling expression of this gene in vascular SMCs. Functional studies on the BTEB2 promoter coupled to a luciferase reporter gene demonstrated activation of the promoter by PMA and basic fibroblast growth factor. Both characterization of DNA-protein complexes in vitro and site-specific mutation analysis of the BTEB2 promoter have defined a 9-bp sequence, 5'-CGCCCGCGC-3', located at -25, as the Egr-1 binding site mediating an induction of the BTEB2 promoter activity by PMA. In addition, we show that this site mediates inducible expression through the mitogen-activated protein kinase pathways. These results indicate that BTEB2 is a target of the early-response gene Egr-1, and mitogen-activated protein kinase pathways directly or indirectly activate BTEB2 expression. Given a rapid induction of Egr-1 on stimulation with growth factors or injury, these findings may represent at least one of the molecular mechanisms underlying phenotypic modulation of smooth muscles after vascular injury.

Animals↗
PubMed1999

Hypoxia induces activation and subcellular translocation of focal adhesion kinase (p125(FAK)) in cultured rat cardiac myocytes.

We previously reported that hypoxia caused rapid activation of RAS/mitogen-activated protein kinase (MAPK) pathway, two other stress-activated MAPK family members, stress-activated protein kinase (SAPK) and p38MAPK, and Src family tyrosine kinases, p60(c-src) and p59(c-fyn) in cultured rat cardiac myocytes. In this study, to elucidate how hypoxia affects adhesive interaction between cardiac myocytes and extracellular matrix (ECM), we investigated the molecular mechanism of the activation of focal adhesion-associated tyrosine kinases p125(FAK) and paxillin. Here, we show that hypoxia induced tyrosine phosphorylation of p125(FAK) and paxillin and that hypoxia-induced activation of p125(FAK) was accompanied by its increased association with adapter proteins Shc and GRB2, and non-receptor type tyrosine kinase p60(c-src). Furthermore, hypoxia caused subcellular translocation of p125(FAK) from perinuclear sites to the focal adhesions. These results strongly suggest that p125(FAK) is one of the most important components in hypoxia-induced intracellular signaling in cardiac myocytes and may play a pivotal role in adhesive interaction between cardiac myocytes and ECM.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Hereditary ceruloplasmin deficiency increases advanced glycation end products in the brain.

We investigated the role of ceruloplasmin in the antioxidative process in the brain in a patient with hereditary ceruloplasmin deficiency (HCD). Immunohistochemistry revealed an accumulation of Nepsilon-(carboxymethyl) lysine (CML) in basal ganglia of the HCD brain. In vitro study disclosed that ceruloplasmin inhibited CML formation from glycated proteins through the reaction of Fe2+ with H2O2 by Fenton reaction. These data suggest that ceruloplasmin plays an important role in the protection of neurons against oxidative stress associated with iron metabolism.

Brain↗
PubMed1999

Polymorphisms in the SOD2 and HLA-DRB1 genes are associated with nonfamilial idiopathic dilated cardiomyopathy in Japanese.

To reveal genetic risk factors of nonfamilial idiopathic cardiomyopathy (IDC) in Japanese, polymorphisms in the SOD2 and HLA-DRB1 genes were investigated in 86 patients and 380 healthy controls. There was a significant excess of homozygotes for the V allele [Val versus Ala (A allele), a polymorphism in the leader peptide of manganese superoxide dismutase at position 16] of the SOD2 gene in the patients compared with the controls (87.2% versus 74.7%, odds ratio = 2.30, p = 0.013, pc < 0.03), and a significant increase in the frequency of HLA-DRB1*1401 in the patients was confirmed (14.0% vs 4.5%, odds ratio = 3.46, p = 0.001, pc < 0.03). A two-locus analysis suggested that these two genetic markers (SOD2-VV genotype and DRB1*1401) may play a synergistic role in controlling the susceptibility to nonfamilial IDC. In addition, processing efficiency of Val-type SOD2 leader peptide in the presence of mitochondria was siginificantly lower than that of the Ala-type by 11 +/- 4%, suggesting that this lower processing efficiency was in part an underlying mechanism of the association between the SOD2-VV genotype and nonfamilial IDC.

Adolescent↗
PubMed1999

Endothelin-1 induces expression of fetal genes through the interleukin-6 family of cytokines in cardiac myocytes.

We here examined the role of the interleukin-6 (IL-6) family of cytokines in endothelin-1 (ET-1)-induced hypertrophic responses using cultured cardiac myocytes of neonatal rats. ET-1 induced expression of IL-6 and leukemia inhibitory factor (LIF) genes. ET-1-induced LIF gene expression was abolished by inhibition of protein kinase C activity. ET-1 activated the promoter of atrial natriuretic peptide and beta-type myosin heavy chain genes through the tyrosine kinase pathway and IL-6 receptor gp130. These results suggest that the IL-6 family of cytokines mediates ET-1-induced expression of some fetal genes in cardiac myocytes.

Animals↗
PubMed1999

BTEB2, a Krüppel-like transcription factor, regulates expression of the SMemb/Nonmuscle myosin heavy chain B (SMemb/NMHC-B) gene.

We have recently characterized the promoter region of the rabbit embryonic smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and identified the 15-bp sequence, designated SE1, located at -105 from the transcriptional start site as an important regulatory element for its transcriptional activity in a smooth muscle cell (SMC) line. In this study, we attempted to isolate cDNA clones encoding for the transcription factors that control the expression of the SMemb gene through binding to this cis-regulatory element. We screened a lambdagt11 cDNA library prepared from C2/2 cells, a rabbit-derived SMC line, by using a radiolabeled concatenated oligonucleotide containing SE1 as a probe. Sequence analysis revealed that one of the cDNA clones corresponds to the rabbit homologue of basic transcriptional element binding protein-2 (BTEB2), which has previously been identified as one of the Krüppel-like transcription factor. Gel mobility shift assays and antibody supershift analyses with nuclear extracts from C2/2 cells indicate that BTEB2 is a major component of nuclear factor:SE1 complexes. Furthermore, a glutathione S-transferase-BTEB2 fusion protein binds to the SE1 in a sequence-specific manner. In support of the functionality of BTEB2 binding, basal promoter activity and BTEB2-induced transcriptional activation were markedly attenuated by the disruption of the SE1. In adult rabbit tissues, BTEB2 mRNA was most highly expressed in intestine, urinary bladder, and uterus. BTEB2 mRNA levels were downregulated in rabbit aorta during normal development. Moreover, immunohistochemical analysis indicated a marked induction of BTEB2 protein in the neointimal SMC after balloon injury in rat aorta. These results suggest that BTEB2 mediates the transcriptional regulation of the SMemb/NMHC-B gene and possibly plays a role in regulating gene expression during phenotypic modulation of vascular SMC.

Age Factors↗
PubMed1999

A Na+-H+ exchange inhibitor (SM-20550) protects from microvascular deterioration and myocardial injury after reperfusion.

Na+-H+ exchange inhibitors may reduce myocardial damage after reperfusion. However, their effects on microvascular deterioration are not known. We examined the potency of a novel Na+-H+ exchange inhibitor, SM-20550 [ N-(Aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonate], and its effects on microvascular damage after reperfusion. In an in vitro study, the Na+-H+ exchange inhibiting activity of SM-20550 was about 10 times greater than that of ethylisopropyl amiloride. In in vivo experiments, we occluded the left circumflex coronary artery in 29 dogs for 2 h and then reperfused for 5 h. SM-20550 was administered either before ischemia (n = 11) or before reperfusion (n = 7). Another 11 dogs served as controls. We found that SM-20550 not only improved coronary vasodilator responses to acetylcholine and adenosine after reperfusion, but also reduced infarct size (P < 0.01). Intramyocardial bleeding, which should reflect microvascular damage, was not found in dogs with SM-20550 treatment. Infarct size was correlated inversely with collateral blood flow in control (both, P < 0.01) but not in SM-20550-treated animals. Furthermore, SM-20550 significantly suppressed ventricular fibrillation during both ischemia and reperfusion. These results suggest that protective effects of Na+-H+ exchange inhibitors on reperfused myocardium are due at least in part to microvascular protection.

Acetylcholine↗
PubMed1999

Membrane proteins of human erythrocytes are modified by advanced glycation end products during aging in the circulation.

Recent immunological studies demonstrated that proteins in vivo in several diseases are subjected to post-translational modification by advanced glycation end products (AGEs), suggesting a potential role of AGEs in aging and age-enhanced disease processes such as diabetic complications, atherosclerosis and Alzheimer's disease. Nvarepsilon-(Carboxymethyl)lysine (CML) is one of the major AGE-structures demonstrated in vivo so far. In the present study, membrane proteins from young erythrocyte population were compared with those from senescent erythrocytes separated from the same individual in their CML-contents using a monoclonal antibody for CML (6D12). SDS-polyacrylamide gel electrophoresis and subsequent Western blot showed that 6D12 bound to the band 1, 2, 3, 4.2, 5, 6 and 7 proteins from senescent erythrocytes, but not to those from young erythrocytes. Furthermore, quantitative estimation of the reactivity of 6D12 to these erythrocyte membranes by ELISA showed that the reactivity of 6D12 to senescent erythrocyte membranes was 3- to 6-fold higher than that of young erythrocyte membranes. These results indicate that membrane proteins of circulating erythrocytes undergo CML-modification, and the modified proteins accumulated in an age-dependent manner during the life span of erythrocytes.

Aging↗
PubMed1999

Receptors for proteins modified by advanced glycation endproducts (AGE)--their functional role in atherosclerosis.

Long-term incubation of proteins with glucose leads, through the formation of early stage products such as Schiff base and Amadori rearrangement products, to the formation of advanced glycation end products (AGE). Recent studies of AGE-structures as well as the receptor for AGE-proteins (AGE-receptors) have emphasized the involvement of protein modification by AGE in aging and age-enhanced disease processes. Immunohistochemical analyses of human atherosclerotic lesions using a monoclonal anti-AGE antibody have demonstrated diffuse extracellular AGE-deposition as well as dense intracellular AGE-deposition in macrophage- and vascular smooth muscle cell (SMC)-derived foam cells. In vitro experiments using both CHO cells overexpressing macrophage scavenger receptor-A (MSR-A) and peritoneal macrophages from MSR-A-knockout mice have shown that the MSR-A plays a major role in endocytic uptake of AGE-proteins by macrophages. Furthermore, in vitro experiments with rabbit arterial SMCs demonstrated a novel AGE-receptor mediating endocytosis of AGE-proteins. These in vivo and in vitro experiments suggest that AGE-proteins formed extracellularly in atherosclerotic lesions are endocytosed by macrophages through MSR-A in the early stage, and by SMCs through the novel AGE-receptor in the advanced stage, implicating functional contribution of the AGE-receptor-mediated interaction of AGE-proteins with these cells to atherosclerotic processes in arterial walls.

Animals↗
PubMed1999

Tumor necrosis factor-alpha decreases the phosphorylation levels of phospholamban and troponin I in spontaneously beating rat neonatal cardiac myocytes.

The tumor necrosis factor (TNF) alpha level is elevated in patients with advanced heart failure, and the phosphorylation of contractile regulatory proteins is reduced in the human heart. We hypothesized that TNFalpha affects the phosphorylation of proteins involved in regulating contraction; phospholamban (PLB), myosin light chain 2 (MLC2) and troponin I (TnI). Spontaneously beating rat neonatal cardiac myocytes, prelabelled with [32P]orthophosphate, were treated with TNFalpha for 30 min, and stimulated with isoproterenol for 5 min. 32P-labelled myofibrillar proteins were isolated by 15% SDS-PAGE. Baseline phosphorylation levels of PLB, TnI and an unknown 23kDa phosphoprotein were decreased by TNFalpha in a dose-dependent manner. Moreover, TNFalpha attenuated the phosphorylation levels of PLB and TnI increased by a concentration of 0.01 microM isoproterenol, but not by 1 microM of isoproterenol. Although TNFalpha had no effect on the cAMP content or cAMP-dependent protein kinase activity in the presence or absence of isoproterenol, an inverse relationship was observed between the concentration of TNFalpha and the cGMP content in cardiac myocytes, and treatment with TNFalpha resulted in a concentration-dependent increase in type 2A protein phosphatase activity. The observation that TNFalpha decreases phosphorylation levels of PLB and TnI in cardiac myocytes suggests that the reduction of these protein phosphorylation levels is partially responsible for alterations of intracellular Ca2+-cycling and the force of contraction in TNF alpha-treated cardiac myocytes. Furthermore, TNFalpha reduces myocyte contraction and protein phosphorylation states possibly via cAMP-independent mechanisms, at least in part, by the activation of type 2A protein phosphatase.

Animals↗
PubMed1999

Advanced glycation endproducts are deposited in neuronal hyaline inclusions: a study on familial amyotrophic lateral sclerosis with superoxide dismutase-1 mutation.

To determine the role of advanced glycation endproducts (AGE) in the pathogenesis of familial amyotrophic lateral sclerosis (ALS) with superoxide dismutase-1 (SOD1) mutation, we investigated the immunohistochemical localization of N(epsilon)-carboxymethyl-lysine (CML), one of the major AGE structures, in spinal cords from three familial ALS patients with a heterozygous Ala to Val substitution at codon 4 in the gene for SOD1. Neuronal hyaline inclusions (NHIs), the abnormal structures seen in some of the remaining lower motor neurons of familial ALS patients with SOD1 mutation, were intensely stained by a monoclonal antibody specific for CML in contrast to the only weakly stained cytoplasm. Immunoelectron microscopy depicted the CML determinants restricted to the granule-associated thick linear structures that mainly compose the NHIs. The NHIs were also recognized by antibodies to SOD1, phosphorylated neurofilament protein and ubiquitin. No focal collection of either CML or SOD1 was found in neurons of the control individuals. Our results indicate that CML is a component of the NHIs of familial ALS patients with SOD1 mutation, and suggest that the CML formation may be mediated by protein glycoxidation or lipid peroxidation in the presence of oxidative stress from mutant SOD1, in association with motor neuron degeneration.

Adult↗
PubMed1999

Induction of smooth muscle cells in the fibrous capsule of human hepatocellular carcinoma but not in the septa of hepatic cirrhosis.

We examined the expression of smooth muscle cytoskeleton in spindle-shaped cells in the capsule of hepatocellular carcinoma (HCC) and the septa of liver cirrhosis (LC). Serial sections of livers resected from 11 patients were stained with monoclonal antibodies against vimentin, desmin, smooth muscle actin (1A4, HHF35, CGA7) and smooth muscle myosin heavy chain isoforms (SM1, SM2). Capsular spindle-shaped cells exhibited a cytoskeletal feature indicative of intermediately differentiated smooth muscle cells. Computer-assisted morphometry revealed that the proportions of 1A4-, HHF35-, CGA7- and SM1- positive areas to vimentin-positive area were 88.0+/-11.0%, 50.8+/-17.4%, 25.3+/-16.4% and 19.4+/-12.4% (n=11) in main tumours and 86.6+/-9.4%, 50.9+/-18.7%, 21.1+/-12.3% and 17.6+/-9.7% (n=12) in daughter tumours, indicating that spindle-shaped cells are heterogeneous in cytoskeletal expression. Septal spindle-shaped cells in LC lacked the cytoskeletal proteins specific to differentiated smooth muscle cells (CGA7, SM1, SM2 and desmin). Electron microscopically, capsular spindle-shaped cells contained more microfilaments and less rough endoplasmic reticulum than do septal cells. Intermediately differentiated smooth muscle cells are induced in the capsule of HCC but not in the septa of LC, suggesting a role for stromal interaction by tumour cells in the induction of smooth muscle cells.

Actins↗
PubMed1999

"White coat effect" induced by therapist's presence during speech therapy for stroke rehabilitation: a single case study.

The excessive pressor response triggered in patients by an alerting reaction to a doctor's presence has been termed the "white coat effect." A 68-year-old man with verbal apraxia after multiple lacunar infarctions was referred to the hospital for speech rehabilitation. He experienced difficulty in talking with the speech therapist during therapy sessions but not when talking with his friends or family. Because the therapist's presence was stressful to the patient, it was considered that his anxiety might produce an excessive increase in blood pressure. Blood pressure monitoring was performed during 2 separate days of speech therapies consisting of two sessions each. In one session, therapy was directed by the therapist; in the other, therapy was self-directed. The therapist-directed approach substantially increased both systolic and diastolic blood pressures, whereas the self-directed therapy slightly increased only systolic pressure. It was concluded that the excessive pressor response seen in this patient during therapist-directed speech therapy resulted from the white coat effect induced by the therapist's presence.

Aged↗
PubMed1999

Increased advanced glycation end products in atherosclerotic lesions of patients with end-stage renal disease.

Although advanced glycation end products (AGEs) are increased in the serum and tissues of patients with end-stage renal disease, little is known about the role of AGEs in atherogenesis. We therefore carried out an immunohistochemical study on the accumulation of AGEs and apolipoprotein B in the human aortas of diabetic and nondiabetic subjects with end-stage renal disease. The atherosclerotic lesions included diffuse intimal thickening, fatty streaks and atherosclerotic plaque. We used antibodies against two different epitopes of AGE structures, i.e. an Nepsilon-(carboxymethyl)lysine-protein adduct (CML) and a structure(s) other than CML (nonCML). The area that was positive for an antigen as a percentage of the total area (%Ar) was determined morphometrically, using an NIH-image program. In diffuse intimal thickening, atherosclerotic plaque and tunica media, the %Ar of CML and nonCML was significantly greater in diabetic or nondiabetic subjects with end-stage renal disease than in control subjects without end-stage renal disease. In fatty streaks, the %Ar of nonCML was significantly greater in nondiabetic subjects with end-stage renal disease than in control subjects, while no difference in the %Ar of CML was found between the subjects with or without end-stage renal disease. Nondiabetic subjects with end-stage renal disease showed a significantly increased %Ar of apolipoprotein B in fatty streaks and atherosclerotic plaque than the control subjects. The %Ar of CML and nonCML significantly correlated with the duration of hemodialysis in diffuse intimal thickening and atherosclerotic plaque of subjects with end-stage renal disease, but not in fatty streaks. On the other hand, the %Ar was not related to the duration of diabetes in any of the lesions in the diabetic subjects with end-stage renal disease. In diffuse intimal thickening and atherosclerotic plaque, subjects with end-stage renal disease showed a significant correlation between the %Ar of apolipoprotein B and AGEs (CML and nonCML), as well as their immunohistochemical colocalization. These results suggest that impaired AGE clearance may cause the increased accumulation of AGEs in the aortic wall of subjects with end-stage renal disease, thus resulting in the rapid progression of atherosclerosis. The accumulation of AGEs may be related to an enhanced LDL deposition in atherosclerotic lesions of subjects with end-stage renal disease.

Aged↗