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Although α-synuclein (SNCA) is a well-known pathological molecule involved in synucleinopathy in neurons, its physiological roles remain largely unknown. We reported that serum SNCA levels have a close inverse correlation with blood pressure and age, which indicates the involvement of SNCA in age-related endothelial dysfunction. Therefore, this study aimed to elucidate the molecular functions of SNCA in the endothelium. We confirmed that SNCA was expressed in and secreted from endothelial cells (ECs). Exogenous treatment with recombinant SNCA (rSNCA) activated the Akt-eNOS axis and increased nitric oxide production in ECs. Treatment with rSNCA also suppressed TNF-α- and palmitic acid-induced NF-κB activation, leading to the suppression of VCAM-1 upregulation and restoration of eNOS downregulation in ECs. As for endogenous SNCA expression, replicative senescence resulted in the attenuation of SNCA expression in cultured ECs, similar to the effects of physiological aging on mice aortas. The siRNA-mediated silencing of SNCA consistently resulted in senescent phenotypes, such as eNOS downregulation, increased β-gal activity, decreased Sirt1 expression, and increased p53 expression, in ECs. Ex vivo assessment of endothelial functions using aortic rings revealed impaired endothelium-dependent acetylcholine-induced relaxation in SNCA knockout (KO) mice. Furthermore, SNCA KO mice, especially those on a high-fat diet, displayed elevated blood pressure compared with wild-type mice; this could be eNOS dysfunction-dependent because of the lower difference caused by L-NAME administration. These results indicate that exogenous and endogenous SNCA in ECs might physiologically maintain vascular integrity, and age-related endothelial dysfunction might be partially ascribed to loss-of-function of SNCA in ECs.

Angiotensin converting enzyme-2 (ACE2) is a multifunctional transmembrane protein recently recognised as the entry receptor of the virus causing COVID-19. In the renin–angiotensin system (RAS), ACE2 cleaves angiotensin II (Ang II) into angiotensin 1-7 (Ang 1-7), which is considered to exert cellular responses to counteract the activation of the RAS primarily through a receptor, Mas, in multiple organs including skeletal muscle. Previous studies have provided abundant evidence suggesting that Ang 1-7 modulates multiple signalling pathways leading to protection from pathological muscle remodelling and muscle insulin resistance. In contrast, there is relatively little evidence to support the protective role of ACE2 in skeletal muscle. The potential contribution of endogenous ACE2 to the regulation of Ang 1-7-mediated protection of these muscle pathologies is discussed in this review. Recent studies have suggested that ACE2 protects against ageing-associated muscle wasting (sarcopenia) through its function to modulate molecules outside of the RAS. Thus, the potential association of sarcopenia with ACE2 and the associated molecules outside of RAS is also presented herein. Further, we introduce the transcriptional regulation of muscle ACE2 by drugs or exercise, and briefly discuss the potential role of ACE2 in the development of COVID-19.

While adipose tissue is required to maintain glucose metabolism, excessive calorie intake induces obesity via mechanisms including accelerated proliferation and differentiation of preadipocytes, leading to insulin resistance. Here, we investigated the role of myoferlin (MYOF), a ferlin family protein, in regulating glucose metabolism by mainly focusing on its unknown role in adipose tissue. Whereas young MYOF knockout (KO) mice on a normal diet showed aggravated glucose tolerance and insulin sensitivity, those on a high-fat diet (HFD) showed preserved glucose tolerance with an attenuated gain of body weight, reduced visceral fat deposits, and less severe fatty liver. The Adipose MYOF expression was reduced by aging but was restored by an HFD along with the retained expression of NFAT transcription factors. Loss-of-function of MYOF in preadipocytes suppressed proliferation and differentiation into mature adipocytes along with the decreased expression of genes involved in adipogenesis. The MYOF expression in preadipocytes was reduced with differentiation. Attenuated obesity in MYOF KO mice on an HFD was also accompanied with increased oxygen consumption by an unidentified mechanism and with reduced adipose inflammation due to less inflammatory macrophages. These insights suggest that the multifunctional roles of MYOF involve the regulation of preadipocyte function and affect glucose metabolism bidirectionally depending on consumed calories.

Background The activation of AT2 (angiotensin II type 2 receptor ) and Mas receptor by angiotensin II and angiotensin‐(1‐7), respectively, is the primary process that counteracts activation of the canonical renin‐angiotensin system (RAS). Although inhibition of canonical RAS could delay the progression of physiological aging, we recently reported that deletion of Mas had no impact on the aging process in mice. Here, we used male mice with a deletion of only AT2 or a double deletion of AT2 and Mas to clarify whether these receptors contribute to the aging process in a complementary manner, primarily by focusing on aging‐related muscle weakness. Methods and Results Serial changes in grip strength of these mice up to 24 months of age showed that AT2/Mas knockout mice, but not AT2 knockout mice, had significantly weaker grip strength than wild‐type mice from the age of 18 months. AT2/Mas knockout mice exhibited larger sizes, but smaller numbers and increased frequency of central nucleation (a marker of aged muscle) of single skeletal muscle fibers than AT2 knockout mice. Canonical RAS‐associated genes, inflammation‐associated genes, and senescence‐associated genes were highly expressed in skeletal muscles of AT2/Mas knockout mice. Muscle angiotensin II content increased in AT2/Mas knockout mice. Conclusions Double deletion of AT2 and Mas in mice exaggerated aging‐associated muscle weakness, accompanied by signatures of activated RAS, inflammation, and aging in skeletal muscles. Because aging‐associated phenotypes were absent in single deletions of the receptors, AT2 and Mas could complement each other in preventing local activation of RAS during aging.

The receptor for advanced glycation end-products (RAGE) and the G protein-coupled angiotensin II (AngII) type I receptor (AT1) play a central role in cardiovascular diseases. It was recently reported that RAGE modifies AngII-mediated AT1 activation via the membrane oligomeric complex of the two receptors. In this study, we investigated the presence of the different directional crosstalk in this phenomenon, that is, the RAGE/AT1 complex plays a role in the signal transduction pathway of RAGE ligands. We generated Chinese hamster ovary (CHO) cells stably expressing RAGE and AT1, mutated AT1, or AT2 receptor. The activation of two types of G protein α-subunit, Gq and Gi, was estimated through the accumulation of inositol monophosphate and the inhibition of forskolin-induced cAMP production, respectively. Rat kidney epithelial cells were used to assess RAGE ligand-induced cellular responses. We determined that RAGE ligands activated Gi, but not Gq, only in cells expressing RAGE and wildtype AT1. The activation was inhibited by an AT1 blocker (ARB) as well as a RAGE inhibitor. ARBs inhibited RAGE ligand-induced ERK phosphorylation, NF-κB activation, and epithelial–mesenchymal transition of rat renal epithelial cells. Our findings suggest that the activation of AT1 plays a central role in RAGE-mediated cellular responses and elucidate the role of a novel molecular mechanism in the development of cardiovascular diseases.

In the early phase of the Coronavirus disease 2019 (COVID‐19) pandemic, it was postulated that the renin‐angiotensin‐system inhibitors (RASi) increase the infection risk. This was primarily based on numerous reports, which stated that the RASi could increase the organ Angiotensin‐converting enzyme 2 (ACE2), the receptor of Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), in rodents. RASi can theoretically antagonize the potential influence of angiotensin II (Ang II) on ACE2. However, while Ang II decreases the ACE2 levels in cultured cells, there is little evidence that supports this phenomenon in living animals. In this study, we tested whether Ang II or Ang II combined with its antagonist would alter the ACE2 and other molecules associated with the infection of SARS‐CoV‐2. Male C57BL6/J mice were administered vehicle, Ang II (400 ng/kg/min), or Ang II with losartan (10 mg/kg/min) for 2 weeks. ACE2 knockout mice were used as a negative control for the ACE2 assay. We found that both Ang II, which elevated blood pressure by 30 mm Hg, and Ang II with losartan, had no effect on the expression or protein activity of ACE2 in the lung, left ventricle, kidney, and ileum. Likewise, these interventions had no effect on the expression of Transmembrane Protease Serine 2 (TMPRSS2) and Furin, proteases that facilitate the virus‐cell fusion, and the expression or activity of Tumor Necrosis Factor α‐Convertase (TACE) that cleaves cell‐surface ACE2. Collectively, physiological concentrations of Ang II do not modulate the molecules associated with SARS‐CoV‐2 infection. These results support the recent observational studies suggesting that the use of RASi is not a risk factor for COVID‐19.

Inhibition of the renin-angiotensin system (RAS) has been shown to alleviate muscle atrophy both under pathological conditions and during physiological aging. We recently reported that the deletion of angiotensin converting enzyme 2 (ACE2), which converts Angiotensin II to Angiotensin-(1-7) in mice, leads to the early manifestation of aging-associated muscle weakness along with the increased expression of p16INK4a, a senescence-associated gene, and increased central nuclei in the tibialis anterior (TA) muscle in middle age. As ACE2 is multifunctional and functions beyond its role in the RAS, we investigated whether activation of the RAS primarily contributes to muscle weakness in ACE2 knockout (KO) mice by comparing these mice to Tsukuba hypertensive (TH) mice that overproduce human angiotensin II. The grip strength of young (6 months) and middle-aged (15 months) TH mice was consistently lower than that of wild-type mice at the same ages. Middle-aged TH mice were continuously lean with extremely reduced adiposity. Central nuclei in the gastrocnemius (GM) muscle were increased in ACE2KO mice, while no apparent morphological change was observed in the GM muscles of TH mice. Increased expression of p16INK4a along with alterations in the expression of several sarcopenia-associated genes were observed in the GM muscles of ACE2KO mice but not TH mice. These findings suggest that chronic overactivation of the RAS does not primarily contribute to the early aging phenotypes of skeletal muscle in ACE2KO mice.

The angiotensin-converting enzyme 2 (ACE2)-angiotensin 1-7 (A1-7)-A1-7 receptor (Mas) axis plays a protective role in the renin–angiotensin system (RAS). We recently found that ACE2 knockout (ACE2KO) mice exhibit earlier aging-associated muscle weakness, and that A1-7 alleviates muscle weakness in aging mice. In the present study, we investigated the role of the A1-7-Mas pathway in the effect of ACE2 on physiological aging. Male wild-type, ACE2KO, and Mas knockout (MasKO) mice were subjected to periodical grip strength measurement, followed by administration of A1-7 or vehicle for 4 weeks at 24 months of age. ACE2KO mice exhibited decreased grip strength after 6 months of age, while grip strength of MasKO mice was similar to that of wild-type mice. A1-7 improved grip strength in ACE2KO and wild-type mice, but not in MasKO mice. Muscle fibre size was smaller in ACE2KO mice than that in wild-type and MasKO mice, and increased with A1-7 in ACE2KO and WT mice, but not in MasKO mice. Centrally nucleated fibres (CNFs) and expression of the senescence-associated gene p16INK4a in skeletal muscles were enhanced only in ACE2KO mice and were not altered by A1-7. ACE2KO mice, but not MasKO mice, exhibited thinning of peripheral fat along with increased adipose expression of p16INK4a. A1-7 significantly increased bone volume in wild-type and ACE2KO mice, but not in MasKO mice. Our findings suggest that the impact of ACE2 on physiological aging does not depend on the endogenous production of A1-7 by ACE2, while overactivation of the A1-7-Mas pathway could alleviate sarcopenia and osteoporosis in aged mice.

Sacubitril/valsartan was shown to attenuate the development of cardiac hypertrophy with enhanced blood pressure reduction compared with valsartan alone in animal models. We investigated whether a low-dose sacubitril/valsartan has blood pressure-independent effects on cardiac hypertrophy and pulmonary edema using a rat model of hypertension and obesity.In plan 1, male SHR/NDmcr-cp rats fed normal or phase-increased high salt were treated with vehicle, 6-mg/kg sacubitril/valsartan or 3-mg/kg valsartan, for 6 months. In plan 2, after high-salt loading for 6 months, drugs were administered for 4 months. Antihypertensive effects of the 2 drugs were similar during all study periods. In plan 1 with normal salt, there were no differences between treatments in the left ventricle weight/body weight (BW), or lung weight/BW as an index of cardiac hypertrophy or pulmonary edema, respectively. These indexes were smaller in high-salt-fed rats with sacubitril/valsartan than vehicle. In plan 2, both indexes did not differ between vehicle and sacubitril/valsartan. Ventricle weight/BW was lower in valsartan than sacubitril/valsartan. In plan 2, gene markers of cardiac dysfunction were upregulated by sacubitril/valsartan compared with the other groups.Low-dose sacubitril/valsartan may have different effects depending on the stage of cardiac hypertrophy in rats.

There has been a growing interest in the association between the number of teeth and dietary intake in older populations. However, people around the age of 80 y have frequently lost most of their teeth, and dental prostheses replacing the missing teeth play an important role in masticatory function. Therefore, masticatory function cannot be evaluated by the number of teeth alone. The occlusal force of the complete dental arches is an index of masticatory function, reflecting not only the number of teeth, but the effect of removable dentures. The purpose of this cross-sectional study was to determine the relative importance of the number of teeth and occlusal force in association with dietary intake in 80-y-old Japanese people. This study included 760 community-dwelling Japanese people aged 79 y to 81 y. The authors measured bilateral maximal occlusal force in the intercuspal position using pressure-sensitive sheets. Removable denture wearers kept their dentures in place during the measurements. Energy-adjusted food groups and nutrient intake during the preceding month were assessed by a brief self-administered diet history questionnaire. The authors assessed linear trends in food and nutrient intake in relation to the number of teeth and occlusal force after adjusting for gender and socioeconomic status (education level, financial status, family structure, resident area and BMI). P values of < 0.05 were considered to be statistically significant. The authors found that the number of teeth was not associated with the energy-adjusted intake of any food group examined. In contrast, a decline in occlusal force was significantly associated with a lower intake of vegetables, fish and shellfish, protein, polyunsaturated fatty acids, dietary fiber and most vitamins and minerals ( P for trend < 0.05). We conclude that food and nutrient intake was more closely associated with occlusal force than the number of teeth in community-dwelling Japanese people aged 79 y to 81 y. Knowledge Transfer Statement: This cross-sectional study of older Japanese people showed that, after controlling for considerable covariates, occlusal force rather than the number of teeth is positively associated with energy-adjusted intake of vegetables, fish and shellfish, protein, polyunsaturated fatty acids, dietary fiber and most of vitamins and minerals. This means that reduced occlusal force may unconsciously lead older people toward a habitual unhealthy dietary intake. Older people have frequently lost most of their teeth and require prosthetics to restore masticatory function. Bilateral occlusal force is therefore a better measure of masticatory function than the number of remaining teeth. Our findings suggest that prosthetic rehabilitation is a significant factor in the prevention and management of chronic diseases and frailty through better dietary intake in older populations.

Recent longitudinal studies have shown the influence of multiple tooth loss on cognitive impairment, and earlier studies suggested that periodontal disease was related to cognitive decline. Tooth loss is associated with reduced masticatory function, which may affect stimulation of the central nervous system and dietary intake. Although some studies have reported a relationship between chewing ability and cognitive function, no studies have examined this area in terms of objective oral function. The aim of this study was to examine the association of occlusal force with cognitive decline in the preclinical stage among older people with higher-level functional capacity. This cross-sectional study for community-dwelling older people living in urban and rural areas in Japan examined 994 persons in the 70-y group (age range, 69–71 y) and 968 persons in the 80-y group (age range, 79–81 y). Retention of higher-level competence was defined according to the Tokyo Metropolitan Institute of Gerontology Index of Competence. Cognitive function was measured with the Japanese version of the Montreal Cognitive Assessment (MoCA-J). Oral status and function were assessed by the number of remaining teeth, periodontal pocket depth, and maximal occlusal force. Associations between the MoCA-J score and occlusal force were examined by bivariate and multivariate analysis. Approximately one-half of the participants retained higher-level functional capacity and were included in the analysis. Multiple regression analysis showed that occlusal force was significantly related to cognitive function after controlling for possible predictors including age, sex, socioeconomic status, medical condition, and handgrip strength. The number of remaining teeth and periodontal pocket depth were not significantly associated with cognitive function. Among community-dwelling older people with retained competence, maximal occlusal force was positively associated with their cognitive function. These results suggest that oral function might be a predictor for preclinical cognitive decline. Knowledge Transfer Statement: Multiple regression analysis showed that occlusal force was significantly related to cognition after controlling for possible predictors including handgrip strength as an indicator of general muscle strength, suggesting the independence of oral function. The number of remaining teeth did not have this association. The majority of older people have lost teeth and have received prosthodontic treatment, and their occlusal force is determined not only by the number of remaining teeth but also by prosthetic rehabilitation. These results can be used by clinicians focusing on prevention of tooth loss among the entire population, as well as to encourage partially edentulous and fully edentulous patients to restore their oral function with prostheses in order to eliminate a possible risk factor for cognitive impairment.

Cardiovascular disease is one of the leading causes of death in the elderly, and novel therapeutic targets against atherogenesis are urgent. The initiation of atherosclerotic changes of monocyte adhesion on the vascular endothelium and subsequent foam cell formation are noteworthy pathophysiologies when searching for strategies to prevent the progression of age-related atherosclerosis. We report the significance of the deubiquitinating enzyme cylindromatosis (CYLD) in vascular remodeling by interference with inflammatory responses regulated by NF-κB signaling. The purpose of this study was to elucidate the pathological functions of CYLD in the early phase of atherogenesis associated with aging. Treatment with inflammatory cytokines induced endogenous CYLD in aortic endothelial cells (HAECs) and THP-1 cells. siRNA-mediated CYLD silencing led to enhanced monocyte adhesion along with increased adhesion molecules in HAECs treated with TNFα. In siRNA-mediated CYLD silenced RAW 264.7 macrophages treated with oxidized LDL (oxLDL), augmented lipid accumulation was observed, along with increased expression of the class A macrophage scavenger receptor (SR-A), lectin-like oxidized LDL receptor-1 (LOX-1), CD36, fatty acid binding protein 4 (FABP4), the cholesterol ester synthase acyl-CoA cholesterol acyltransferase (ACAT1), MCP-1, and IL-1β and decreased expression of scavenger receptor class B type I (SR-BI). Intriguingly, CYLD gene expression was significantly reduced in bone marrow-derived macrophages of aged mice compared that of young mice, as well as in senescent HAECs compared with young cells. These findings suggest that age-related attenuation of CYLD expression in endothelial cells (ECs) and macrophages triggers the initiation of age-related atherogenesis by exacerbating monocyte adhesion on the endothelium and foam cell formation. CYLD in the vasculature may be a novel therapeutic target, especially in the early preventive intervention against the initiation of age-related atherogenesis.

The angiotensin II type 1 receptor (AT1) is a 7-transmembrane domain GPCR that when activated by its ligand angiotensin II, generates signaling events promoting vascular dysfunction and the development of cardiovascular disease. Here, we show that the single-transmembrane oxidized LDL (oxLDL) receptor (LOX-1) resides in proximity to AT1 on cell-surface membranes and that binding of oxLDL to LOX-1 can allosterically activate AT1-dependent signaling events. oxLDL-induced signaling events in human vascular endothelial cells were abolished by knockdown of AT1 and inhibited by AT1 blockade (ARB). oxLDL increased cytosolic G protein by 350% in Chinese hamster ovary (CHO) cells with genetically induced expression of AT1 and LOX-1, whereas little increase was observed in CHO cells expressing only LOX-1. Immunoprecipitation and in situ proximity ligation assay (PLA) assays in CHO cells revealed the presence of cell-surface complexes involving LOX-1 and AT1. Chimeric analysis showed that oxLDL-induced AT1 signaling events are mediated via interactions between the intracellular domain of LOX-1 and AT1 that activate AT1. oxLDL-induced impairment of endothelium-dependent vascular relaxation of vascular ring from mouse thoracic aorta was abolished by ARB or genetic deletion of AT1. These findings reveal a novel pathway for AT1 activation and suggest a new mechanism whereby oxLDL may be promoting risk for cardiovascular disease.

The conventional forelimb grip strength test is a widely used method to assess skeletal muscle function in rodents; in this study, we modified this method to improve its variability and consistency. The modified test had lower variability among trials and days than the conventional test in young C57BL6 mice, especially by improving the variabilities in male. The modified test was more sensitive than the conventional test to detect a difference in motor function between female and male mice, or between young and old male mice. When the modified test was performed on male mice during the aging process, reduction of grip strength manifested between 18 and 24 months of age at the group level and at the individual level. The modified test was similar to the conventional test in detecting skeletal muscle dysfunction in young male dystrophic mice. Thus, the modified forelimb grip strength test, with its improved validity and reliability may be an ideal substitute for the conventional method.

This cross-sectional study aimed to investigate the association of periodontal status with occlusal force and food acceptability. We hypothesised that mastication deteriorated with reduced periodontal support, even when posterior occlusal contacts with natural teeth were maintained and the patients remained clinically asymptomatic. Participants were 482 independently living 69-71-year-olds, classified as Eichner's group A, having no mobile teeth and no periodontal symptoms. The periodontal probing depth (PPD) and restoration status of each tooth were examined. Occlusal force in the intercuspal position was measured with pressure-sensitive films. Food acceptability was evaluated from the difficulty experienced in chewing apples, grilled beef, and hard rice crackers. Multivariate regression analysis was performed to investigate the association of periodontal status with occlusal force and food acceptability. A P-value of

ACE type 2 (ACE2) functions as a negative regulator of the renin-angiotensin system by cleaving angiotensin II (AII) into angiotensin 1–7 (A1–7). This study assessed the role of endogenous ACE2 in maintaining insulin sensitivity. Twelve-week-old male ACE2 knockout (ACE2KO) mice had normal insulin sensitivities when fed a standard diet. AII infusion or a high-fat, high-sucrose (HFHS) diet impaired glucose tolerance and insulin sensitivity more severely in ACE2KO mice than in their wild-type (WT) littermates. The strain difference in glucose tolerance was not eliminated by an AII receptor type 1 (AT1) blocker but was eradicated by A1–7 or an AT1 blocker combined with the A1–7 inhibitor (A779). The expression of GLUT4 and a transcriptional factor, myocyte enhancer factor (MEF) 2A, was dramatically reduced in the skeletal muscles of the standard diet–fed ACE2KO mice. The expression of GLUT4 and MEF2A was increased by A1–7 in ACE2KO mice and decreased by A779 in WT mice. A1–7 enhanced upregulation of MEF2A and GLUT4 during differentiation of myoblast cells. In conclusion, ACE2 protects against high-calorie diet–induced insulin resistance in mice. This mechanism may involve the transcriptional regulation of GLUT4 via an A1–7–dependent pathway.

The development of atherosclerosis is associated with disturbances in mitochondrial function that impair effective adenosine triphosphate (ATP) production, increase generation of superoxide and induce subsequent apoptosis in vascular smooth muscle cells (VSMCs). As peroxisome proliferator-activated receptor gamma (PPARγ) has a potentially important role in the regulation of mitochondrial metabolism, we studied effects of the partial PPARγ agonist and angiotensin receptor blocker telmisartan, on mitochondria-related cellular responses in VSMC. In human VSMC, telmisartan increased ATP levels and activation of mitochondrial complex II, succinate dehydrogenase, reduced the release of H2O2 and attenuated H2O2-induced increases in caspase 3/7 activity, a marker of cellular apoptosis. Eprosartan, an angiotensin II receptor blocker that lacks the ability to activate PPARγ, had no effect on these mitochondria-related cellular responses in VSMC. Studies in PPARγ-deficient VSMC revealed that the effects of telmisartan on mitochondrial function were largely independent of PPARγ although the presence of PPARγ modulated effects of telmisartan on H2O2 levels. These findings demonstrate that telmisartan can have significant effects on mitochondrial metabolism in VSMC that are potentially relevant to the pathogenesis of cardiovascular disease and that involve more than just angiotensin receptor blockade and activation of PPARγ.