Angiotensin Converting Enzyme Regulates Cell Proliferation and Migration

Background The angiotensin-I converting enzyme (ACE) plays a central role in the renin-angiotensin system, acting by converting the hormone angiotensin-I to the active peptide angiotensin-II (Ang-II). More recently, ACE was shown to act as a receptor for Ang-II, and its expression level was demonstrated to be higher in melanoma cells compared to their normal counterparts. However, the function that ACE plays as an Ang-II receptor in melanoma cells has not been defined yet. Aim Therefore, our aim was to examine the role of ACE in tumor cell proliferation and migration. Results We found that upon binding to ACE, Ang-II internalizes with a faster onset compared to the binding of Ang-II to its classical AT1 receptor. We also found that the complex Ang-II/ACE translocates to the nucleus, through a clathrin-mediated process, triggering a transient nuclear Ca2+ signal. In silico studies revealed a possible interaction site between ACE and phospholipase C (PLC), and experimental results in CHO cells, demonstrated that the beta 3 isoform of PLC is the one involved in the Ca2+ signals induced by Ang-II/ACE interaction. Further studies in melanoma cells (TM-5) showed that Ang-II induced cell proliferation through ACE activation, an event that could be inhibited either by ACE inhibitor (Lisinopril) or by the silencing of ACE. In addition, we found that stimulation of ACE by Ang-II caused the melanoma cells to migrate, at least in part due to decreased vinculin expression, a focal adhesion structural protein. Conclusion ACE activation regulates melanoma cell proliferation and migration.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)INCT Nanocarbono - UFMG (Brazil)Univ Fed Minas Gerais, Dept Physiol & Biophys, Belo Horizonte, MG, BrazilUniv Fed Sao Joao del Rei, Dept Nat Sci, Sao Joao Del Rei, MG, BrazilUniv Fed Ceara, Dept Phys, Fortaleza, CE, BrazilUniv Fed Sao Paulo, Dept Biophys, Sao Paulo, SP, BrazilUniv Fed Minas Gerais, Dept Phys, Belo Horizonte, MG, BrazilUniv Fed Minas Gerais, Dept Morphol, Belo Horizonte, MG, BrazilDepartment of Biophysics, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, BrazilWeb of Scienc

Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: In an era of shifting global agendas and expanded emphasis on non-communicable diseases and injuries along with communicable diseases, sound evidence on trends by cause at the national level is essential. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) provides a systematic scientific assessment of published, publicly available, and contributed data on incidence, prevalence, and mortality for a mutually exclusive and collectively exhaustive list of diseases and injuries. Methods: GBD estimates incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) due to 369 diseases and injuries, for two sexes, and for 204 countries and territories. Input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service use, air pollution monitors, satellite imaging, disease notifications, and other sources. Cause-specific death rates and cause fractions were calculated using the Cause of Death Ensemble model and spatiotemporal Gaussian process regression. Cause-specific deaths were adjusted to match the total all-cause deaths calculated as part of the GBD population, fertility, and mortality estimates. Deaths were multiplied by standard life expectancy at each age to calculate YLLs. A Bayesian meta-regression modelling tool, DisMod-MR 2.1, was used to ensure consistency between incidence, prevalence, remission, excess mortality, and cause-specific mortality for most causes. Prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases and injuries to calculate YLDs. We considered results in the context of the Socio-demographic Index (SDI), a composite indicator of income per capita, years of schooling, and fertility rate in females younger than 25 years. Uncertainty intervals (UIs) were generated for every metric using the 25th and 975th ordered 1000 draw values of the posterior distribution. Findings: Global health has steadily improved over the past 30 years as measured by age-standardised DALY rates. After taking into account population growth and ageing, the absolute number of DALYs has remained stable. Since 2010, the pace of decline in global age-standardised DALY rates has accelerated in age groups younger than 50 years compared with the 1990–2010 time period, with the greatest annualised rate of decline occurring in the 0–9-year age group. Six infectious diseases were among the top ten causes of DALYs in children younger than 10 years in 2019: lower respiratory infections (ranked second), diarrhoeal diseases (third), malaria (fifth), meningitis (sixth), whooping cough (ninth), and sexually transmitted infections (which, in this age group, is fully accounted for by congenital syphilis; ranked tenth). In adolescents aged 10–24 years, three injury causes were among the top causes of DALYs: road injuries (ranked first), self-harm (third), and interpersonal violence (fifth). Five of the causes that were in the top ten for ages 10–24 years were also in the top ten in the 25–49-year age group: road injuries (ranked first), HIV/AIDS (second), low back pain (fourth), headache disorders (fifth), and depressive disorders (sixth). In 2019, ischaemic heart disease and stroke were the top-ranked causes of DALYs in both the 50–74-year and 75-years-and-older age groups. Since 1990, there has been a marked shift towards a greater proportion of burden due to YLDs from non-communicable diseases and injuries. In 2019, there were 11 countries where non-communicable disease and injury YLDs constituted more than half of all disease burden. Decreases in age-standardised DALY rates have accelerated over the past decade in countries at the lower end of the SDI range, while improvements have started to stagnate or even reverse in countries with higher SDI. Interpretation: As disability becomes an increasingly large component of disease burden and a larger component of health expenditure, greater research and developm nt investment is needed to identify new, more effective intervention strategies. With a rapidly ageing global population, the demands on health services to deal with disabling outcomes, which increase with age, will require policy makers to anticipate these changes. The mix of universal and more geographically specific influences on health reinforces the need for regular reporting on population health in detail and by underlying cause to help decision makers to identify success stories of disease control to emulate, as well as opportunities to improve. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

Angiotensin Converting Enzyme Regulates Cell Proliferation and Migration.

The angiotensin-I converting enzyme (ACE) plays a central role in the renin-angiotensin system, acting by converting the hormone angiotensin-I to the active peptide angiotensin-II (Ang-II). More recently, ACE was shown to act as a receptor for Ang-II, and its expression level was demonstrated to be higher in melanoma cells compared to their normal counterparts. However, the function that ACE plays as an Ang-II receptor in melanoma cells has not been defined yet.Therefore, our aim was to examine the role of ACE in tumor cell proliferation and migration.We found that upon binding to ACE, Ang-II internalizes with a faster onset compared to the binding of Ang-II to its classical AT1 receptor. We also found that the complex Ang-II/ACE translocates to the nucleus, through a clathrin-mediated process, triggering a transient nuclear Ca2+ signal. In silico studies revealed a possible interaction site between ACE and phospholipase C (PLC), and experimental results in CHO cells, demonstrated that the β3 isoform of PLC is the one involved in the Ca2+ signals induced by Ang-II/ACE interaction. Further studies in melanoma cells (TM-5) showed that Ang-II induced cell proliferation through ACE activation, an event that could be inhibited either by ACE inhibitor (Lisinopril) or by the silencing of ACE. In addition, we found that stimulation of ACE by Ang-II caused the melanoma cells to migrate, at least in part due to decreased vinculin expression, a focal adhesion structural protein.ACE activation regulates melanoma cell proliferation and migration

ACE silencing inhibits the proliferative effect of Ang-II in melanoma cells.

<p>(A) Western bot (upper panel) to confirm the silencing of ACE and densitometry analysis (bottom panel). Mean ± S.E.M, n = 8. (***p<0.01 compared to respective columns). (B) BrDU uptake in Tm5 cells silenced for ACE and stimulated for 24 hours with Ang-II (1μM), showing a decrease in BrDU incorporation in the absence of ACE. Mean ± S.E.M., n = 12 (*p<0.05; ns = non-significant).</p

Ang-II promotes cellular migration and reduces focal adhesion formation in melanoma cells.

<p>(A-B) Wound healing assay using TM-5 cells stimulated with Ang-II (1μM). C) Representative confocal images of TM-5 cells double-labeled with vinculin (green) and phalloidin (red). Scale bar = 10 μm. (D-E) Quantification of the focal adhesion formation. Mean ± S.E.M., n = 6 (* p<0.05).</p

Ang-II induces ACE translocation to the nucleus.

<p>(A) Internalization of AT₁ and ACE in the presence of 4 nM ³H-Ang-II. Data are shown as mean from three independent experiments, each performed in duplicate. (B) CHO-ACE and CHO-AT₁ cells present the same relative protein level of each respective receptor. (Values are mean ± S.E.M, *p<0.05, n = 63 individual experiments). (C) Representative confocal images of internalized Ang-II-FITC (1 μM) in CHO-ACE and CHO-AT₁ cells after 30 seconds of Ang-II stimulation. DAPI (blue) and Wheat Germ Agglutinin (red), scale bar = 10 μm. On the right, quantification of internalized Ang-II-FITC is presented. Values are mean ± S.E.M, *p<0.05, n = 6. (D) Representative confocal images of unstimulated CHO-ACE and CHO-AT1 cells, labeled for DAPI and WGA. (E) Immunolocalization of ACE after stimulation with Ang-II (1μM), for the indicated times. ACE is shown in green, actin filaments in red, and nucleus in blue (DAPI). Right panel represents a 3D reconstruction of CHO-ACE cell after 15 minutes of incubation with Ang-II (1μM). Scale bar = 10μm. (F) Western blotting of nuclear and non-nuclear protein fractions from CHO-ACE cells, before (control) and after Ang-II (1 μM) stimulation for the indicated times. Histone-3 and GAPDH were used to shown the purification of nuclear and non-nuclear protein fractions, respectively. (G) Densitometry analysis of the western blot. Values are mean ± S.E.M., n = 3 (*** p<0.01).</p

Cell proliferation induced by Ang-II/ACE involves clathrin- mediated internalization process.

<p>(A) Cell growth assay of CHO-ACE cells 12, 24 and 48 hours after stimulation with Ang-II (1μM), triplicate in 3 individual experiments. (B) Western blot to confirm the silencing of clathrin (upper panel) and densitometry analysis (bottom panel). Mean ± S.E.M., n = 5 (* p<0.05). (C) BrDU incorporation is decreased in CHO-ACE cells transfected with siRNA-Cla (Clathrin) and stimulated with Ang-II (1μM). Mean ± S.E.M., n = 6 (* p<0.05).</p