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.

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.

Serum albumin/globulin ratio is associated with cognitive function in community-dwelling older people: The Septuagenarians, Octogenarians, Nonagenarians Investigation with Centenarians study.

AIM: The objective of this study was to clarify the relationship between cognitive function and the serum albumin/globulin ratio (A/G ratio) in community-dwelling Japanese older adults. METHODS: Randomly extracted residents in both urban and rural parts of Japan were enrolled in this study. A total of 1827 participants with a mean age of 70 or 80 years were recruited. A venue survey method was carried out with comprehensive studies, including interviews, blood collection, physical examination and cognitive function tests. RESULTS: Univariate analysis showed a significant positive correlation between the total Japanese version of the Montreal Cognitive Assessment score and the serum A/G ratio at the age of 70 and 80 years, in which better cognitive function was associated with a high serum A/G ratio. Multiple regression analysis with the total Japanese version of the Montreal Cognitive Assessment score as the dependent variable showed that the serum albumin level, serum globulin level, serum A/G ratio, C-reactive protein, years of formal education and sex were related to the Japanese version of the Montreal Cognitive Assessment total score at the age of 70 years, and that the serum albumin level, serum globulin level, serum A/G ratio, C-reactive protein, years of formal education and stroke were related at the age of 80 years. The serum A/G ratio showed a better correlation than the serum globulin levels at the age of 70 and 80 years (70 years: ß = 0.131 vs -0.111, 80 years: ß = 0.108 vs -0.071). CONCLUSIONS: We found a correlation between cognitive function and the serum A/G ratio in community-dwelling older people, suggesting that nutritional status and chronic inflammation might influence cognitive function. Geriatr Gerontol Int 2019; 19: 967-971.

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.

Influence of antihypertensive drugs in the subtype diagnosis of primary aldosteronism by adrenal venous sampling.

OBJECTIVES: Because of the influence on the renin-angiotensin-aldosterone system, it is recommended to avoid, if possible, the use of angiotensin-converting-enzyme inhibitors, angiotensin II type 1 receptor blockers, diuretics, ß-blockers, and mineralocorticoid receptor antagonists during the diagnostic period of primary aldosteronism. A laterality index more than 4 in adrenocorticotropic hormone (ACTH)-stimulated adrenal venous sampling (ACTH-AVS) is a widely used classification of the unilateral subtype that can benefit from adrenalectomy. Here, we revealed clinical features of patients taking drugs that could affect the primary aldosteronism diagnosis (DAPD) and investigated whether the classification with laterality index more than 4 in ACTH-AVS is applicable to these patients. PATIENTS AND METHODS: Using a large database of primary aldosteronism patients in Japan, we analyzed 2122 patients with successful ACTH-AVS. RESULTS: Patients who received any DAPD (n = 209) showed higher prevalence of comorbidity burdens and took more antihypertensive drugs compared with patients without DAPD. In patients taking DAPD, those with laterality index more than 4 had a higher prevalence of hypokalemia, a higher aldosterone-to-renin ratio and a higher prevalence of adrenal mass than those with laterality index of 4 or less. Adrenalectomy was performed in 76% patients with laterality index more than 4 and 20% with laterality index of 4 or less. Patients who underwent adrenalectomy showed biochemical cure in 89% with laterality index more than 4 and 50% with laterality index of 4 or less (P = 0.001). Multivariate regression analysis showed that laterality index more than 4 was an independent predictor of a biochemical cure. Biochemical cure rate in patients with laterality index more than 4 was consistently high, irrespective of the potential effect of individual DAPD on laterality index. CONCLUSION: Our findings suggest that in primary aldosteronism patients to whom DAPD were administrated due to severe clinical features, laterality index more than 4 in ACTH-AVS could accurately predict a biochemical cure after adrenalectomy.

Association between long-term care and chronic and lifestyle-related disease modified by social profiles in community-dwelling people aged 80 and 90; SONIC study.

BACKGROUND: Chronic and lifestyle-related diseases and social status were reported to be associated with long-term care (LTC). The social factors should be treated as social sub-groups of which characteristics show social profiles. However, few previous studies considered that. The present study aimed to investigate the associations between LTC and chronic and lifestyle-related diseases, and whether the associations were modified by the social sub-groups in the community-dwelling elderly. METHOD: A cross-sectional study was conducted among 1004 community-dwelling participants aged 80 and 90. LTC was used as the outcome. Chronic and lifestyle-related diseases (i.e., stroke, heart disease, joint pain, osteoporosis, lung disease, cancer, hypertension, dyslipidemia, and diabetes) were used as the predictors. Education, household income, residential area, and support environment were analyzed by latent class analysis (LCA) to derive social profiles. We obtained odds ratios (ORs) of LTC from those diseases and tested interactions between those diseases and the social profiles by logistic regression analyses. RESULT: The participants were categorized into two sub-groups of social factors (n = 675 and 329) by LCA. Logistic regression analyses showed ORs (95% CI) of LTC were 4.69 (2.49, 8.71) from stroke, 2.22 (1.46, 3.38) from joint pain, 1.99 (1.22, 3.25) from osteoporosis, and 2.05 (1.22, 3.40) from cancer adjusting for the social sub-groups. There were no significant interactions between the social subgroups and those diseases in relation to LTC except for osteoporosis. CONCLUSION: The associations between LTC and chronic and lifestyle-related diseases were significant with adjusting for the social sub-groups, and not modified by that except osteoporosis.

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.

Clinical Characteristics and Postoperative Outcomes of Primary Aldosteronism in the Elderly.

Context: Primary aldosteronism (PA) in the elderly has increased in importance in association with population aging. Objective: To investigate the characteristics and outcomes of elderly patients with PA undergoing adrenalectomy. Patients and Methods: Using a database of patients with PA who underwent adrenal venous sampling (AVS), we compared elderly patients (≥65 years old) with nonelderly patients (<65 years old) in terms of characteristics, subtype classification in ACTH-stimulated AVS, and outcomes after adrenalectomy. Results: The elderly group had a higher prevalence of comorbidities than the nonelderly group. The proportion of the unilateral subtype [defined as a lateralization index (LI) >4] was comparable between the age groups. In patients who received adrenalectomy, biochemical cure was comparable between the groups, whereas persistent hypertension was more common in the elderly group. The prevalences of hyperkalemia and renal impairment (chronic kidney disease stage 3b or higher) were higher in the elderly group. Multiple regression analysis showed that the duration of hypertension predicted persistent hypertension and hyperkalemia and that preoperative estimated glomerular filtration rate predicted renal impairment in the elderly group. LI >4 in AVS was an independent predictor of biochemical cure after adrenalectomy in the elderly group but not in the nonelderly group. Age was negatively associated with biochemical cure in patients with LI ≤4. Conclusion: Adrenalectomy contributes to biochemical improvement in elderly patients if determined in accordance with AVS. The treatment strategy should be determined considering the high postoperative incidence of persistent hypertension and hyperkalemia in elderly patients with a long history of hypertension or renal impairment in those with reduced renal function.

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.

High plasma adiponectin levels are associated with frailty in a general old-old population: The Septuagenarians, Octogenarians, Nonagenarians Investigation with Centenarians study.

AIM: The objective of the present study was to investigate the association between frailty and plasma adiponectin levels in a general population of Japanese older adults. METHODS: The volunteer older adults, aged approximately 83 years, were recruited randomly from a general population in the Japanese Septuagenarians, Octogenarians, Nonagenarians Investigation with Centenarians study. We used the modified Cardiovascular Health Study criteria to assess the frailty status of the study participants. The study participants were classified as non-frail, pre-frail and frail according to their physical activities. We compared plasma adiponectin levels among these three groups and applied a multivariate logistic regression analysis including plasma adiponectin levels to clarify the factors associated with frailty status in the cross-sectional design. RESULTS: The mean age of the participants was 83.1 ± 0.9 years, and 51.8% were men. The frailty index was available to assess 353 participants, of whom 24.6% were classified as non-frail, 62.3% as prefrail and 13.0% as frail. The log-transformed plasma adiponectin levels increased stepwise in the following order: non-frail, pre-frail and frail. A multivariate logistic regression analysis showed that higher plasma adiponectin levels, a higher estimated glomerular filtration rate and lower hemoglobin levels were independent determinants for pre-frail/frail status compared with non-frail status. CONCLUSIONS: The present study showed that higher plasma adiponectin levels were associated with frailty status in older Japanese adults in the general population. Further longitudinal study is essential to clarify the role of plasma adiponectin in the progression of frailty. Geriatr Gerontol Int 2018; 18: 839-846.

Longitudinal association of hypertension and diabetes mellitus with cognitive functioning in a general 70-year-old population: the SONIC study.

Both hypertension and diabetes in middle-aged individuals have been suggested to be predictive indicators of cognitive decline. However, the association of hypertension, diabetes and their combination with cognitive functioning is still controversial in older people. The purpose of this study was to investigate the association between cognitive decline and hypertension, diabetes, and their combination in 70-year-old people based on a 3-year longitudinal analysis. Four hundred and fifty-four people aged 70 (±1) years who participated in the Japanese longitudinal cohort study of Septuagenarians, Octogenarians and Nonagenarians Investigation with Centenarians (SONIC) were recruited randomly from a general population and were monitored for 3 years. The data, including most of the demographics, cognitive functioning measured by the Montreal Cognitive Assessment Japanese version (MoCA-J), blood pressure, blood chemistry and other medical histories, were collected at baseline and during the follow-up. The prevalence of hypertension noted in the follow-up survey was significantly higher than than noted at baseline. The mean MoCA-J score at follow-up was not significantly different from the score obtained at baseline. However, the participants with diabetes, especially combined with hypertension at baseline, had significantly lower MoCA-J scores than those without lifestyle-related diseases. The combination of hypertension and diabetes was still a significant risk factor for cognitive decline, considering the MoCA-J scores obtained during the follow-up after adjustments at baseline, relative to sex, body mass index, dyslipidemia, smoking, excessive alcohol intake, antihypertensive treatment and education level (ß=-0.14; P<0.01). Our findings indicate that diabetes and the combination of hypertension and diabetes are clear risk factors for future cognitive decline in elderly individuals who are 70 years of age.

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.

Factors associated with the severity of obstructive sleep apnea in older adults.

AIM: Epidemiological studies have shown that severe obstructive sleep apnea (OSA) is associated with higher mortality when compared with mild to moderate OSA. Because aging is a well-known risk factor for OSA, we aimed to elucidate the underlying factors associated with the severity of OSA in elderly patients. METHODS: Patients who underwent polysomnography were divided into the non-elderly group (aged <65 years; n = 44) and the elderly group (aged ≥65 years; n = 46). The severity of OSA was determined by the apnea hypopnea index (AHI), and each group was subdivided into two groups: mild to moderate OSA (5 < AHI < 30) and severe OSA (AHI ≥30) . In the elderly group, geriatric assessments to evaluate physical and neuropsychiatric function were carried out. RESULTS: All patients had OSA as diagnosed by an AHI >5. Whereas body mass index was positively correlated with AHI in both groups, age was correlated with AHI only in the elderly group. Body mass index and age were higher in severe OSA than mild to moderate OSA in the elderly group. Unexpectedly, no significant difference was observed in physical strength, cognitive function, apathy scale, depression scale or activities of daily living between mild to moderate OSA and severe OSA in the elderly group. Binary logistic regression analysis showed that male sex, body mass index and aging were independent risk factors of severe OSA in the elderly group. CONCLUSIONS: Our findings suggest that aging increases the severity of OSA in elderly patients, even if they are physically active and neuropsychiatrically unimpaired. Geriatr Gerontol Int 2017; 17: 614-621.

Differences in the association between high blood pressure and cognitive functioning among the general Japanese population aged 70 and 80 years: The SONIC study.

High blood pressure in middle age (up to 64 years) has been proposed as a predictive indicator of dementia. However, the association between hypertension and the cognitive functioning is controversial in older age groups. The aim of this study was to investigate this association in 70-80-year-old participants in the Japanese study of Septuagenarians, Octogenarians and Nonagenarians Investigation with Centenarians (SONIC). Participants aged 70 (±1) and 80 (±1) years (n=1000 and 973, respectively) were randomly recruited from the general population in Japan. Cognitive functioning was measured by the Montreal Cognitive Assessment. Blood pressure and other medical and social variables were analyzed by multiple regression analyses. High systolic blood pressure (SBP) was significantly correlated with a reduced cognitive functioning only in participants aged 70 years. Additionally, this correlation became more marked in participants with uncontrolled blood pressure at age 70 years. In contrast, SBP was not significantly correlated with the cognitive functioning at age 80 years. Nutritional status indicators such as serum albumin and frequency of going outdoors were significantly associated with cognitive functioning at age 80 years. Our findings indicate that high SBP has a significant role in cognitive functioning at age 70 years; however, blood pressure is less important as a risk factor for cognitive decline at age 80 years.

Oxidized LDL (oxLDL) activates the angiotensin II type 1 receptor by binding to the lectin-like oxLDL receptor.

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.

Loss of ACE2 exaggerates high-calorie diet-induced insulin resistance by reduction of GLUT4 in mice.

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.

Telmisartan modulates mitochondrial function in vascular smooth muscle cells.

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.

The counterregulating role of ACE2 and ACE2-mediated angiotensin 1-7 signaling against angiotensin II stimulation in vascular cells.

To clarify the role of endogenous angiotensin (Ang)-converting enzyme 2 (ACE2) and its cleavage product, Ang 1-7, in the atherogenic stimulation of vascular cells, we investigated the effect of pharmacological inhibition of ACE2 and Mas, an Ang 1-7 receptor, on cellular responses against Ang II stimulation. We measured extracellular signal-regulated kinase (ERK) 1/2 phosphorylation by western blot, smooth muscle cell (SMC) proliferation by WST assay and the adhesion of monocytes labeled with PKH67 to endothelial cells (ECs) by fluorescence microplate reader. Cells were pretreated with Ang 1-7, olmesartan (Ang II type 1 receptor (AT1) blocker), DX600 (ACE2 inhibitor), -Ala7-Ang1-7 (D-Ala; Mas antagonist), or combinations of treatments before the application of Ang II. Treatment with Ang II increased phosphorylated ERK 1/2 of SMC and EC, proliferation of SMC and adhesion of monocyte to EC, which were blocked by olmesartan. Pretreatment with DX600 either did not accelerate or only slightly accelerated these cellular responses. However, when Ang II signaling through AT1 was reduced by olmesartan, the additional treatment with DX600 significantly blunted some of the effect of olmesartan. Similarly, pretreatment with D-Ala reduced the inhibitory effect of olmesartan in response to Ang II stimulation. Endogenous ACE2 in vascular cells may contribute to counteracting the Ang II-mediated cellular response partly by upregulating the Ang 1-7 signaling through Mas.

Loss of ACE2 accelerates time-dependent glomerular and tubulointerstitial damage in streptozotocin-induced diabetic mice.

As angiotensin-converting enzyme-2 (ACE2) was identified as a negative regulator of the renin-angiotensin system, there have been many reports concerning its role in several tissues, including the kidney. However, the role of ACE2 during the development of diabetic nephropathy remains undetermined, as previous reports did not necessarily support a protective role against renal injury. Thus, we performed detailed observations of kidneys in ACE2-knockout (ACE2-KO) mice at early (4 weeks) and advanced (18 weeks) stages of diabetes. ACE2-KO and wild-type C57BL/6 mice were rendered diabetic by intraperitoneal injection of streptozotocin. Diabetic ACE2-KO mice showed earlier onset and more severe progression of albuminuria than those did wild-type mice. The elevation of serum creatinine and urea nitrogen levels at 18 weeks of diabetes was more prominent in ACE2-KO mice. Periodic acid-Schiff-stained cross-section of diabetic ACE2-KO mice showed a more severe time-dependent increase in glomerular/tubulointerstitial damage than did that of wild-type mice, confirmed by the immunostaining of alpha-smooth muscle actin, collagen IV and F4-80 antigen. Glomeruli of diabetic ACE2-KO mice showed earlier and more severe decrease in the expression of nephrin, whose degradation is involved in the onset of albuminuria, and more potent increase of vascular endothelial growth factor expression. In addition, treatment with AT1 receptor blocker olmesartan significantly, but not totally, ameliorated the functional and morphological deterioration of diabetic nephropathy in ACE2-KO mice. These results suggest that ACE2 might continuously protect from both glomerular and tubulointerstitial injury during the development of diabetic nephropathy. The renal-protective effect of ACE2 might involve more than just suppressing angiotensin II-mediated AT1 receptor signaling.