Is the anti-aging effect of ACE2 due to its role in the renin-angiotensin system?-Findings from a comparison of the aging phenotypes of ACE2-deficient, Tsukuba hypertensive, and Mas-deficient mice.

Angiotensin converting enzyme 2 (ACE2) functions as an enzyme that produces angiotensin 1-7 (A1-7) from angiotensin II (AII) in the renin-angiotensin system (RAS). We evaluated aging phenotypes, especially skeletal muscle aging, in ACE2 systemically deficient (ACE2 KO) mice and found that ACE2 has an antiaging function. The characteristic aging phenotype observed in ACE2 KO mice was not reproduced in mice deficient in the A1-7 receptor Mas or in Tsukuba hypertensive mice, a model of chronic AII overproduction, suggesting that ACE2 has a RAS-independent antiaging function. In this review, the results we have obtained and related studies on the aging regulatory mechanism mediated by RAS components will be presented and summarized. We evaluated the aging phenotype of ACE2 systemically deficient (ACE2 KO) mice, particularly skeletal muscle aging, and found that ACE2 has an antiaging function. The characteristic aging phenotype observed in ACE2 KO mice was not reproduced in Mas KO mice, angiotensin 1-7 receptor-deficient mice or in Tsukuba hypertensive mice, a model of chronic angiotensin II overproduction, suggesting that the antiaging functions of ACE2 are independent of the renin-angiotensin system (RAS).

The endocytosis of oxidized LDL via the activation of the angiotensin II type 1 receptor.

Arrestin-dependent activation of a G-protein-coupled receptor (GPCR) triggers endocytotic internalization of the receptor complex. We analyzed the interaction between the pattern recognition receptor (PRR) lectin-like oxidized low-density lipoprotein (oxLDL) receptor (LOX-1) and the GPCR angiotensin II type 1 receptor (AT1) to report a hitherto unidentified mechanism whereby internalization of the GPCR mediates cellular endocytosis of the PRR ligand. Using genetically modified Chinese hamster ovary cells, we found that oxLDL activates Gαi but not the Gαq pathway of AT1 in the presence of LOX-1. Endocytosis of the oxLDL-LOX-1 complex through the AT1-ß-arrestin pathway was demonstrated by real-time imaging of the membrane dynamics of LOX-1 and visualization of endocytosis of oxLDL. Finally, this endocytotic pathway involving GPCR kinases (GRKs), ß-arrestin, and clathrin is relevant in accumulating oxLDL in human vascular endothelial cells. Together, our findings indicate that oxLDL activates selective G proteins and ß-arrestin-dependent internalization of AT1, whereby the oxLDL-LOX-1 complex undergoes endocytosis.

Novel properties of myoferlin in glucose metabolism via pathways involving modulation of adipose functions.

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.

Different effects of the deletion of angiotensin converting enzyme 2 and chronic activation of the renin-angiotensin system on muscle weakness in middle-aged mice.

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.

A case of primary aldosteronism with resistant hypertension successfully treated by unilateral adrenalectomy after unsuccessful classification of subtype in adrenal venous sampling.

Despite being an established method to identify the unilateral subtype of primary aldosteronism with an indication of adrenalectomy, adrenal venous sampling sometimes fails primarily due to unsuccessful cannulation to adrenal veins. In such cases, the analysis of clinical findings might help to identify the indication of surgery.

Angiotensin 1-7 alleviates aging-associated muscle weakness and bone loss, but is not associated with accelerated aging in ACE2-knockout 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.

Severity of obstructive sleep apnea is associated with the nocturnal fluctuation of pulse rate, but not with that of blood pressure, in older hypertensive patients receiving calcium channel blockers.

AIM: Obstructive sleep apnea (OSA) is associated with increased variability in nocturnal blood pressure (BP). Calcium channel blockers (CCB) are superior to other classes of antihypertensives in decreasing BP variability. We investigated whether OSA severity is associated with nocturnal BP variability in older hypertensive patients treated with CCB. METHODS: We measured home systolic and diastolic BP and pulse rate (PR) automatically during sleep at an interval of an hour once a week using an electronic sphygmomanometer in 29 hypertensive patients (aged ≥65 years) receiving CCB. We calculated the coefficient of variation (CV) from four consecutive measurements. All patients underwent a home-based portable sleep study. RESULTS: We found no difference in PR, BP or CV of BP between the patients with no-to-mild OSA and with moderate-to-severe OSA, categorized by the respiratory disturbance index (RDI) and 3% oxygen desaturation index (ODI). The CV of PR in patients with moderate-to-severe OSA was higher than the patients with no-to-mild OSA categorized by 3% ODI (P = 0.01). Body mass index was correlated with RDI and 3% ODI (r = 0.56 and 0.43, respectively). The CV of BP did not correlate to RDI or 3% ODI. The CV of PR was positively correlated both with RDI and with 3% ODI (r = 0.41 and 0.42, respectively). CONCLUSIONS: The severity of OSA was associated with PR variability, but not with BP variability, in older patients receiving CCB. Our results suggest the need for future studies to determine whether CCB can suppress the influence of OSA on BP fluctuation during sleep. Geriatr Gerontol Int 2019; 19: 604-610.

Pathophysiological significance of cylindromatosis in the vascular endothelium and macrophages for the initiation of age-related atherogenesis.

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-1ß 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.

Angiotensin-converting enzyme 2 deficiency accelerates and angiotensin 1-7 restores age-related muscle weakness in mice.

BACKGROUND: A pharmacologic strategy for age-related muscle weakness is desired to improve mortality and disability in the elderly. Angiotensin-converting enzyme 2 (ACE2) cleaves angiotensin II into angiotensin 1-7, a peptide known to protect against acute and chronic skeletal muscle injury in rodents. Since physiological aging induces muscle weakness via mechanisms distinct from other muscle disorders, the role of ACE2-angiotensin 1-7 in age-related muscle weakness remains undetermined. Here, we investigated whether deletion of ACE2 alters the development of muscle weakness by aging and whether angiotensin 1-7 reverses muscle weakness in older mice. METHODS: After periodic measurement of grip strength and running distance in male ACE2KO and wild-type mice until 24 months of age, we infused angiotensin 1-7 or vehicle for 4 weeks, and measured grip strength, and excised tissues. Tissues were also excised from younger (3-month-old) and middle-aged (15-month-old) mice. Microarray analysis of RNA was performed using tibialis anterior (TA) muscles from middle-aged mice, and some genes were further tested using RT-PCR. RESULTS: Grip strength of ACE2KO mice was reduced at 6 months and was persistently lower than that of wild-type mice (p < 0.01 at 6, 12, 18, and 24-month-old). Running distance of ACE2KO mice was shorter than that of wild-type mice only at 24 months of age [371 ± 26 vs. 479 ± 24 (m), p < 0.01]. Angiotensin 1-7 improved grip strength in both types of older mice, with larger effects observed in ACE2KO mice (% increase, 3.8 ± 1.5 and 13.3 ± 3.1 in wild type and ACE2KO mice, respectively). Older, but not middle-aged ACE2KO mice had higher oxygen consumption assessed by a metabolic cage than age-matched wild-type mice. Angiotensin 1-7 infusion modestly increased oxygen consumption in older mice. There was no difference in a wheel-running activity or glucose tolerance between ACE2KO and wild-type mice and between mice with vehicle and angiotensin 1-7 infusion. Analysis of TA muscles revealed that p16INK4a, a senescence-associated gene, and central nuclei of myofibers increased in middle-aged, but not younger ACE2KO mice. p16INK4a and central nuclei increased in TA muscles of older wild-type mice, but the differences between ACE2KO and wild-type mice remained significant (p < 0.01). Angiotensin 1-7 did not alter the expression of p16INK4a or central nuclei in TA muscles of both types of mice. Muscle ACE2 expression of wild-type mice was the lowest at middle age (2.6 times lower than younger age, p < 0.05). CONCLUSIONS: Deletion of ACE2 induced the early manifestation of muscle weakness with signatures of muscle senescence. Angiotensin 1-7 improved muscle function in older mice, supporting future application of the peptide or its analogues in the treatment of muscle weakness in the elderly population.

Comparison between L-type and N/L-type calcium channel blockers in the regulation of home blood-pressure variability in elderly hypertensive patients.

Recent studies suggest that L-type calcium channel blockers (CCBs) contribute to reducing blood pressure (BP) variability. We investigated whether inhibition of the N-type calcium channel has an additional effect on BP variability by comparing the effect of L-type and L/N-type CCBs on home BP variability in elderly hypertensive patients. Twenty-six hypertensive patients (≥65 years) were subjected to repeated changes with the administration of amlodipine (L-type CCB) and cilnidipine (L/N-type CCB) every 2 months. They measured the home BP in the morning and evening, and the coefficient of variation (CV) was calculated. We measured the brachial-ankle pulse wave velocity (baPWV) and urinary catecholamine excretion as an index of the arterial stiffness and sympathetic nerve activity, respectively. There was no difference in the effect of both drugs on the CV in the morning and evening, while amlodipine was associated with a modestly higher pulse rate and lower BP than cilnidipine. By comparing individual patient data for the CV with each drug, we found that higher urinary catecholamine excretion was associated with the effectiveness of cilnidipine over amlodipine in the BP variability in the morning, which was not the case in the evening. In contrast, lower baPWV was associated with the effectiveness of amlodipine over cilnidipine on BP variability in the evening. Lower baPWV was also associated with lower BP variability in the evening. Cilnidipine has a similar capacity as amlodipine in reducing home BP variability, but the underlying mechanisms in reducing BP variability may differ.

Modified forelimb grip strength test detects aging-associated physiological decline in skeletal muscle function in male mice.

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.

Improvement of health status evaluated by Arthritis Impact Measurement Scale 2 (AIMS-2) and Short Form-36 (SF-36) in patients with rheumatoid arthritis treated with tocilizumab.

OBJECTIVE: To evaluate the improvement of health status in patients with rheumatoid arthritis (RA) treated with tocilizumab. METHODS: Thirty-nine patients were treated with 8 mg/kg tocilizumab every 4 weeks for 24 weeks. Disease activity was assessed by Clinical Disease Activity Index (CDAI) and Simplified Disease Activity Index (SDAI). Improvement of health status was assessed by Arthritis Impact Measurement Scale 2 (AIMS-2) and Short Form-36 (SF-36). RESULTS: Tocilizumab improved CDAI and SDAI significantly at week 4 compared with at baseline. In the components of AIMS-2, "physical score", "symptom" and "affect" improved significantly at week 4 compared with at baseline, while "social interaction" did not improve significantly during 24 weeks of tocilizumab therapy. Similarly in SF-36, "bodily pain", "general health", "vitality" and "mental health" improved significantly at week 4. The most correlative component of AIMS-2 with CDAI was "symptom", while "social interaction" did not correlate with CDAI during tocilizumab treatment. CONCLUSION: The time-course diversity in improvement of health status should be considered to provide proper healthcare when treated with tocilizumab.