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1.
Lab Invest ; 104(5): 102036, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38408704

RESUMO

Arterioles are key determinants of the total peripheral vascular resistance, which, in turn, is a key determinant of arterial blood pressure. However, the amount of protein available from one isolated human arteriole may be less than 5 µg, making proteomic analysis challenging. In addition, obtaining human arterioles requires manual dissection of unfrozen clinical specimens. This limits its feasibility, especially for powerful multicenter clinical studies in which clinical specimens need to be shipped overnight to a research laboratory for arteriole isolation. We performed a study to address low-input, test overnight tissue storage and develop a reference human arteriolar proteomic profile. In tandem mass tag proteomics, use of a booster channel consisting of human induced pluripotent stem cell-derived endothelial and vascular smooth muscle cells (1:5 ratio) increased the number of proteins detected in a human arteriole segment with a false discovery rate of <0.01 from 1051 to more than 3000. The correlation coefficient of proteomic profile was similar between replicate arterioles isolated freshly, following cold storage, or before and after the cold storage (1-way analysis of variance; P = .60). We built a human arteriolar proteomic profile consisting of 3832 proteins based on the analysis of 12 arteriole samples from 3 subjects. Of 1945 blood pressure-relevant proteins that we curated, 476 (12.5%) were detected in the arteriolar proteome, which was a significant overrepresentation (χ2 test; P < .05). These findings demonstrate that proteomic analysis is feasible with arterioles isolated from human adipose tissue following cold overnight storage and provide a reference human arteriolar proteome profile highly valuable for studies of arteriole-related traits.


Assuntos
Tecido Adiposo , Proteômica , Humanos , Arteríolas/metabolismo , Proteômica/métodos , Tecido Adiposo/metabolismo , Tecido Adiposo/irrigação sanguínea , Proteoma/metabolismo , Proteoma/análise , Feminino , Masculino , Adulto , Pessoa de Meia-Idade
2.
Am J Physiol Heart Circ Physiol ; 326(5): H1279-H1290, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38517225

RESUMO

The circulating milieu, bioactive molecules in the bloodstream, is altered with aging and interfaces constantly with the vasculature. This anatomic juxtaposition suggests that circulating factors may actively modulate arterial function. Here, we developed a novel, translational experimental model that allows for direct interrogation of the influence of the circulating milieu on age-related arterial dysfunction (aortic stiffening and endothelial dysfunction). To do so, we exposed young and old mouse arteries to serum from young and old mice and young and midlife/older (ML/O) adult humans. We found that old mouse and ML/O adult human, but not young, serum stiffened young mouse aortic rings, assessed via elastic modulus (mouse and human serum, P = 0.003 vs. young serum control), and impaired carotid artery endothelial function, assessed by endothelium-dependent dilation (EDD) (mouse serum, P < 0.001; human serum, P = 0.006 vs. young serum control). Furthermore, young mouse and human, but not old, serum reduced aortic elastic modulus (mouse serum, P = 0.009; human serum, P < 0.001 vs. old/MLO serum control) and improved EDD (mouse and human serum, P = 0.015 vs. old/MLO serum control) in old arteries. In human serum-exposed arteries, in vivo arterial function assessed in the human donors correlated with circulating milieu-modulated arterial function in young mouse arteries (aortic stiffness, r = 0.634, P = 0.005; endothelial function, r = 0.609, P = 0.004) and old mouse arteries (aortic stiffness, r = 0.664, P = 0.001; endothelial function, r = 0.637, P = 0.003). This study establishes novel experimental approaches for directly assessing the effects of the circulating milieu on arterial function and implicates changes in the circulating milieu as a mechanism of in vivo arterial aging.NEW & NOTEWORTHY Changes in the circulating milieu with advancing age may be a mechanism underlying age-related arterial dysfunction. Ex vivo exposure of young mouse arteries to the circulating milieu from old mice or midlife/older adults impairs arterial function whereas exposure of old mouse arteries to the circulating milieu from young mice or young adults improves arterial function. These findings establish that the circulating milieu directly influences arterial function with aging.


Assuntos
Envelhecimento , Endotélio Vascular , Camundongos Endogâmicos C57BL , Rigidez Vascular , Vasodilatação , Animais , Humanos , Masculino , Adulto , Pessoa de Meia-Idade , Feminino , Endotélio Vascular/fisiopatologia , Idoso , Fatores Etários , Camundongos , Aorta/fisiopatologia , Artérias Carótidas/fisiopatologia , Adulto Jovem , Módulo de Elasticidade
3.
J Biol Chem ; 298(12): 102620, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36272645

RESUMO

Fission protein 1 (FIS1) and dynamin-related protein 1 (DRP1) were initially described as being evolutionarily conserved for mitochondrial fission, yet in humans the role of FIS1 in this process is unclear and disputed by many. In budding yeast where Fis1p helps to recruit the DRP1 ortholog from the cytoplasm to mitochondria for fission, an N-terminal "arm" of Fis1p is required for function. The yeast Fis1p arm interacts intramolecularly with a conserved tetratricopeptide repeat core and governs in vitro interactions with yeast DRP1. In human FIS1, NMR and X-ray structures show different arm conformations, but its importance for human DRP1 recruitment is unknown. Here, we use molecular dynamics simulations and comparisons to experimental NMR chemical shifts to show the human FIS1 arm can adopt an intramolecular conformation akin to that observed with yeast Fis1p. This finding is further supported through intrinsic tryptophan fluorescence and NMR experiments on human FIS1 with and without the arm. Using NMR, we observed the human FIS1 arm is also sensitive to environmental changes. We reveal the importance of these findings in cellular studies where removal of the FIS1 arm reduces DRP1 recruitment and mitochondrial fission similar to the yeast system. Moreover, we determined that expression of mitophagy adapter TBC1D15 can partially rescue arm-less FIS1 in a manner reminiscent of expression of the adapter Mdv1p in yeast. These findings point to conserved features of FIS1 important for its activity in mitochondrial morphology. More generally, other tetratricopeptide repeat-containing proteins are flanked by disordered arms/tails, suggesting possible common regulatory mechanisms.


Assuntos
Dinaminas , GTP Fosfo-Hidrolases , Proteínas de Membrana , Proteínas Mitocondriais , Humanos , Dinaminas/genética , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Am J Physiol Heart Circ Physiol ; 324(3): H330-H337, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36607795

RESUMO

Despite data showing that estrogen is vasculoprotective in large conduit arteries, hormone therapy (HT) during menopause has not proven to mitigate cardiovascular disease (CVD) risk. Estrogen exposure through prolonged oral contraceptive use and gender-affirming therapy can also increase cis- and trans-females' risk for future CVD, respectively. The microvasculature is a unique vascular bed that when dysfunctional can independently predict future adverse cardiac events; however, studies on the influence of estrogen on human microvessels are limited. Here, we show that isolated human arterioles from females across the life span maintain nitric oxide (NO)-mediated dilation to flow, whereas chronic (16-20 h) exposure to exogenous (100 nM) 17ß-estradiol promotes microvascular endothelial dysfunction in vessels from adult females of <40 and ≥40 yr of age. The damaging effect of estrogen was more dramatic in arterioles from biological males, as they exhibited both endothelial and smooth muscle dysfunction. Furthermore, females of <40 yr have greater endothelial expression of estrogen receptor-ß (ER-ß) and G protein-coupled estrogen receptor (GPER) compared with females of ≥40 yr and males. Estrogen receptor-α (ER-α), the prominent receptor associated with protective effects of estrogen, was identified within the adventitia as opposed to the endothelium across all groups. To our knowledge, this is the first study to report the detrimental effects of estrogen on the human microvasculature and highlights differences in estrogen receptor expression.NEW & NOTEWORTHY Microvascular dysfunction is an independent predictor of adverse cardiac events; however, the effect of estrogen on the human microcirculation represents a critical knowledge gap. To our knowledge, this is the first study to report sex-specific detrimental effects of chronic estrogen on human microvascular reactivity. These findings may offer insight into the increased CVD risk associated with estrogen use in both cis- and trans-females.


Assuntos
Receptores de Estrogênio , Doenças Vasculares , Masculino , Adulto , Feminino , Humanos , Arteríolas/metabolismo , Receptores de Estrogênio/metabolismo , Vasodilatação , Estradiol/farmacologia , Estradiol/metabolismo , Estrogênios/farmacologia , Estrogênios/metabolismo , Doenças Vasculares/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Endotélio Vascular/metabolismo
5.
Mol Ther ; 30(11): 3462-3476, 2022 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-35965413

RESUMO

MicroRNA miR-29 promotes endothelial function in human arterioles in part by targeting LYPLA1 and increasing nitric oxide production. In addition, miR-29 is a master inhibitor of extracellular matrix gene expression, which may attenuate fibrosis but could also weaken tissue structure. The goal of this study was to test whether miR-29 could be developed as an effective, broad-acting, and safe therapeutic. Substantial accumulation of miR-29b and effective knockdown of Lypla1 in several mouse tissues were achieved using a chitosan-packaged, chemically modified miR-29b mimic (miR-29b-CH-NP) injected systemically at 200 µg/kg body weight. miR-29b-CH-NP, injected once every 3 days, significantly attenuated angiotensin II-induced hypertension. In db/db mice, miR-29b-CH-NP treatment for 12 weeks decreased cardiac and renal fibrosis and urinary albuminuria. In uninephrectomized db/db mice, miR-29b-CH-NP treatment for 20 weeks significantly improved myocardial performance index and attenuated proteinuria. miR-29b-CH-NP did not worsen abdominal aortic aneurysm in ApoE knockout mice treated with angiotensin II. miR-29b-CH-NP caused aortic root fibrotic cap thinning in ApoE knockout mice fed a high-cholesterol and high-fat diet but did not worsen the necrotic zone or mortality. In conclusion, systemic delivery of low-dose miR-29b-CH-NP is an effective therapeutic for several forms of cardiovascular and renal disease in mice.


Assuntos
Quitosana , Complicações do Diabetes , Diabetes Mellitus , Hipertensão , MicroRNAs , Camundongos , Humanos , Animais , Angiotensina II/efeitos adversos , MicroRNAs/genética , MicroRNAs/metabolismo , Camundongos Knockout para ApoE , Modelos Animais de Doenças , Hipertensão/genética , Hipertensão/terapia , Fibrose , Camundongos Endogâmicos , Tioléster Hidrolases
6.
Vasc Med ; 26(4): 374-382, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33813968

RESUMO

Sedentary behavior is associated with cardiovascular disease (CVD) and mortality, independent of physical activity. The biological mechanisms underlying these associations are largely unknown. We hypothesized that obese subjects with sedentary desk jobs, when assigned a sit-stand desk, will reduce daily sedentary time, and show improvement in arterial flow-mediated dilation (FMD), an early indicator of CVD. Overweight and obese subjects without known CVD were recruited at our institution and given an adjustable sit-stand desk at work. Activities were quantified with an accelerometer for 7 days at baseline and during the intervention. FMD of the brachial and superficial femoral arteries, fasting lipids, insulin and glucose labs, and anthropometrics were measured at baseline, and 12 and 24 weeks. Repeated one-way ANOVA tests were used to compare measurements over time. Fifteen participants were enrolled (93% female, mean age 40 ± 5 years, mean body mass index [BMI] 33 ± 5). Mean daily sedentary time at work decreased by 90 minutes from baseline (385 ± 49 minutes) to 12 weeks (297 ± 80 minutes, p = 0.002) and 24 weeks (295 ± 127 minutes, p = 0.015). Femoral FMD increased from baseline (4.9 ± 1.7%) to 12 weeks (6.4 ± 2.3%, p = 0.043) and further to 24 weeks (8.1 ± 3.2%, p = 0.009). Significant improvement in fasting triglycerides and insulin resistance occurred. There was no change in brachial FMD, exercise activity, step counts, weight, or BMI. A significant reduction in sedentary time during working hours was identified with utilization of a sit-stand desk and sustained over 24 weeks. Improvements in FMD, triglycerides, and insulin resistance provide insight into mechanisms of adverse health risks associated with sedentary behavior.


Assuntos
Doenças Cardiovasculares , Local de Trabalho , Adulto , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/prevenção & controle , Exercício Físico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/diagnóstico , Comportamento Sedentário
7.
Vasc Med ; 26(6): 595-601, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34286655

RESUMO

INTRODUCTION: Prior data suggest associations between hearing loss, cardiovascular (CV) risk factors, and CV disease. Whether specific hearing loss patterns, including a strial pattern associated with inner ear vascular disease, are associated with systemic endothelial dysfunction and carotid intima-media thickness (IMT) remains unclear. METHODS: We evaluated participants without prevalent CVD in the Framingham Offspring Study who underwent formal audiogram testing and brachial and carotid artery ultrasounds. Audiograms were categorized as normal or as belonging to one of four abnormal patterns: cochlear-conductive, low-sloping, sensorineural, or strial. Endothelial function as measured by brachial artery flow-mediated dilation (FMDmm and FMD%). Internal and common intima-media thicknesses (icIMT and ccIMT, respectively) were compared between audiogram patterns. RESULTS: We studied 1672 participants (mean age 59 years, 57.6% women). The prevalence of each hearing pattern was as follows: 43.7% normal; 20.3% cochlear-conductive; 20.3% sensorineural; 7.7% low-sloping; and 8.0% strial. Strial pattern hearing loss was nearly twice as prevalent (p = 0.001) in those in the highest quartile of ccIMT and nearly 50% higher in those in the highest icIMT quartile (p = 0.04). There were no statistically significant differences between the prevalence of the strial pattern comparing the lowest quartiles of FMDmm and FMD% with the upper three quartiles. Age- and sex-adjusted linear regression models did not show significant associations between the vascular measures and hearing patterns. CONCLUSION: Abnormal hearing patterns were not significantly associated with impaired brachial FMD and increased carotid IMT after adjusting for age and sex effects, which may reflect age and sex-related distributional differences based on hearing loss pattern.


Assuntos
Espessura Intima-Media Carotídea , Vasodilatação , Artéria Braquial/diagnóstico por imagem , Artérias Carótidas/diagnóstico por imagem , Endotélio Vascular , Feminino , Audição , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Ultrassonografia
8.
Circ Res ; 132(2): 182-184, 2023 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-36656969
10.
Circ Res ; 123(9): 1091-1102, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30355158

RESUMO

RATIONALE: A strong association has emerged between the gut microbiome and atherosclerotic disease. Our recent data suggest Lactobacillus plantarum 299v (Lp299v) supplementation reduces infarct size in male rats. Limited human data are available on the impact of Lp299v on the vasculature. OBJECTIVE: To determine whether oral Lp299v supplementation improves vascular endothelial function and reduces systemic inflammation in humans with stable coronary artery disease (CAD). METHODS AND RESULTS: Twenty men with stable CAD consumed a drink containing Lp299v (20 billion CFU) once daily for 6 weeks. After a 4-week washout, subjects were given an option of additionally participating in a 10-day study of oral liquid vancomycin (250 mg QID). Vascular endothelial function was measured by brachial artery flow-mediated dilation. Before and after Lp299v, plasma short-chain fatty acids, trimethylamine oxide, and adipokine levels were measured. Additional plasma samples underwent unbiased metabolomic analyses using liquid chromatography/mass spectroscopy. 16S rRNA sequencing was used to determine changes of the stool microbiome. Arterioles from patients with CAD were obtained, and endothelium-dependent vasodilation was measured by video microscopy after intraluminal incubation with plasma from Lp299v study subjects. Lp299v supplementation improved brachial flow-mediated dilation ( P=0.008) without significant changes in plasma cholesterol profiles, fasting glucose, or body mass index. Vancomycin did not impact flow-mediated dilation. Lp299v supplementation decreased circulating levels of IL (interleukin)-8 ( P=0.01), IL-12 ( P=0.02), and leptin ( P=0.0007) but did not significantly change plasma trimethylamine oxide concentrations ( P=0.27). Plasma propionate ( P=0.004) increased, whereas acetate levels decreased ( P=0.03). Post-Lp299v plasma improved endothelium-dependent vasodilation in resistance arteries from patients with CAD ( P=0.02).16S rRNA analysis showed the Lactobacillus genus was enriched in postprobiotic stool samples without other changes. CONCLUSIONS: Lp299v improved vascular endothelial function and decreased systemic inflammation in men with CAD, independent of changes in traditional risk factors and trimethylamine oxide. Circulating gut-derived metabolites likely account for these improvements and merit further study. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov . Unique identifier: NCT01952834.


Assuntos
Doença da Artéria Coronariana/terapia , Citocinas/sangue , Endotélio Vascular/fisiopatologia , Mediadores da Inflamação/sangue , Lactobacillus plantarum/crescimento & desenvolvimento , Probióticos/administração & dosagem , Vasodilatação , Adipocinas/sangue , Adulto , Idoso , Biomarcadores/sangue , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/microbiologia , Doença da Artéria Coronariana/fisiopatologia , Endotélio Vascular/metabolismo , Ácidos Graxos/sangue , Fezes/microbiologia , Microbioma Gastrointestinal , Humanos , Lactobacillus plantarum/genética , Masculino , Metilaminas/sangue , Pessoa de Meia-Idade , Projetos Piloto , Probióticos/efeitos adversos , Fatores de Tempo , Resultado do Tratamento
11.
BMC Geriatr ; 20(1): 162, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32375676

RESUMO

BACKGROUND: In patients with permanent pacemakers (PPM), physical activity (PA) can be monitored using embedded accelerometers to measure pacemaker detected active hours (PDAH), a strong predictor of mortality. We examined the impact of a PA Counseling (PAC) intervention on increasing activity as measured by PDAH and daily step counts. METHODS: Thirteen patients (average age 80 ± 6 years, 84.6% women) with implanted Medtronic PPMs with a ≤ 2 PDAH daily average were included in this study. Patients were randomized to Usual Care (UC, N = 6) or a Physical Activity Counseling Intervention (PACI, N = 7) groups. Step count and PDAH data were obtained at baseline, following a 12-week intervention, then 12 weeks after intervention completion. Data were analyzed using independent t-tests, Pearson's r, chi-square, and general linear models for repeated measures. RESULTS: PDAH significantly differed by time point for all subject combined (P = 0.01) but not by study group. Subjects with baseline gait speeds of > 0.8 m/sec were responsible for the increases in PDAH observed. Step counts did not differ over time in the entire cohort or by study group. Step count and PDAH significantly correlated at baseline (r = 0.60, P = 0.03). This correlation disappeared by week 12. CONCLUSION(S): PDAH can be used to monitor PA and PA interventions and may be superior to hip-worn pedometers in detecting activity. A significant increase in PA, regardless of treatment group, suggests that patient awareness of the ability to monitor PA through a PPM increases PA in these patients, particularly in patients with gait speeds of < 0.8 m/sec. TRIAL REGISTRATION: ClincalTrials.gov NCT03052829. Date of Registration: 2/14/2017.


Assuntos
Actigrafia , Marca-Passo Artificial , Idoso , Idoso de 80 Anos ou mais , Aconselhamento , Exercício Físico , Feminino , Humanos , Masculino , Caminhada
12.
Circulation ; 137(18): e495-e522, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29618598

RESUMO

Physical inactivity is one of the most prevalent major health risk factors, with 8 in 10 US adults not meeting aerobic and muscle-strengthening guidelines, and is associated with a high burden of cardiovascular disease. Improving and maintaining recommended levels of physical activity leads to reductions in metabolic, hemodynamic, functional, body composition, and epigenetic risk factors for noncommunicable chronic diseases. Physical activity also has a significant role, in many cases comparable or superior to drug interventions, in the prevention and management of >40 conditions such as diabetes mellitus, cancer, cardiovascular disease, obesity, depression, Alzheimer disease, and arthritis. Whereas most of the modifiable cardiovascular disease risk factors included in the American Heart Association's My Life Check - Life's Simple 7 are evaluated routinely in clinical practice (glucose and lipid profiles, blood pressure, obesity, and smoking), physical activity is typically not assessed. The purpose of this statement is to provide a comprehensive review of the evidence on the feasibility, validity, and effectiveness of assessing and promoting physical activity in healthcare settings for adult patients. It also adds concrete recommendations for healthcare systems, clinical and community care providers, fitness professionals, the technology industry, and other stakeholders in order to catalyze increased adoption of physical activity assessment and promotion in healthcare settings and to contribute to meeting the American Heart Association's 2020 Impact Goals.


Assuntos
Doenças Cardiovasculares/prevenção & controle , Exercício Físico , Promoção da Saúde , Estilo de Vida Saudável , Comportamento de Redução do Risco , American Heart Association , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/fisiopatologia , Nível de Saúde , Humanos , Prognóstico , Fatores de Proteção , Fatores de Risco , Comportamento Sedentário , Estados Unidos/epidemiologia
13.
Cardiovasc Diabetol ; 18(1): 148, 2019 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-31706320

RESUMO

BACKGROUND: Excessive reactive oxygen species from endothelial mitochondria in type 2 diabetes individuals (T2DM) may occur through multiple related mechanisms, including production of mitochondrial reactive oxygen species (mtROS), inner mitochondrial membrane (Δψm) hyperpolarization, changes in mitochondrial mass and membrane composition, and fission of the mitochondrial networks. Inner mitochondrial membrane proteins uncoupling protein-2 (UCP2) and prohibitin (PHB) can favorably impact mtROS and mitochondrial membrane potential (Δψm). Circulating levels of UCP2 and PHB could potentially serve as biomarker surrogates for vascular health in patients with and without T2DM. METHODS: Plasma samples and data from a total of 107 individuals with (N = 52) and without T2DM (N = 55) were included in this study. Brachial artery flow mediated dilation (FMD) was measured by ultrasound. ELISA was performed to measure serum concentrations of PHB1 and UCP2. Mitochondrial membrane potential was measured from isolated leukocytes using JC-1 dye. RESULTS: Serum UCP2 levels were significantly lower in T2DM subjects compared to control subjects (3.01 ± 0.34 vs. 4.11 ± 0.41 ng/mL, P = 0.04). There were no significant differences in levels of serum PHB. UCP2 levels significantly and positively correlated with FMDmm (r = 0.30, P = 0.03) in T2DM subjects only and remained significant after multivariable adjustment. Within T2DM subjects, serum PHB levels were significantly and negatively correlated with UCP2 levels (ρ = - 0.35, P = 0.03). CONCLUSION: Circulating UCP2 levels are lower in T2DM patients and correlate with endothelium-dependent vasodilation in conduit vessels. UCP2 could be biomarker surrogate for overall vascular health in patients with T2DM and merits additional investigation.


Assuntos
Artéria Braquial/fisiopatologia , Diabetes Mellitus Tipo 2/sangue , Leucócitos/metabolismo , Mitocôndrias/metabolismo , Proteínas Repressoras/sangue , Proteína Desacopladora 2/sangue , Vasodilatação , Adulto , Idoso , Biomarcadores/sangue , Artéria Braquial/diagnóstico por imagem , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Humanos , Masculino , Potencial da Membrana Mitocondrial , Pessoa de Meia-Idade , Projetos Piloto , Proibitinas
14.
Anal Biochem ; 552: 81-99, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29505779

RESUMO

Mitochondria are found in a variety of shapes, from small round punctate structures to a highly interconnected web. This morphological diversity is important for function, but complicates quantification. Consequently, early quantification efforts relied on various qualitative descriptors that understandably reduce the complexity of the network leading to challenges in consistency across the field. Recent application of state-of-the-art computational tools have resulted in more quantitative approaches. This prospective highlights the implementation of MitoGraph, an open-source image analysis platform for measuring mitochondrial morphology initially optimized for use with Saccharomyces cerevisiae. Here Mitograph was assessed on five different mammalian cells types, all of which were accurately segmented by MitoGraph analysis. MitoGraph also successfully differentiated between distinct mitochondrial morphologies that ranged from entirely fragmented to hyper-elongated. General recommendations are also provided for confocal imaging of labeled mitochondria (using mito-YFP, MitoTracker dyes and immunostaining parameters). Widespread adoption of MitoGraph will help achieve a long-sought goal of consistent and reproducible quantification of mitochondrial morphology.


Assuntos
Mitocôndrias/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Humanos , Proteínas Luminescentes/metabolismo , Transfecção
15.
Am J Physiol Heart Circ Physiol ; 312(3): H515-H527, 2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-27923790

RESUMO

Intensive glycemic regulation has resulted in an increased incidence of hypoglycemia. Hypoglycemic burden correlates with adverse cardiovascular complications and contributes acutely and chronically to endothelial dysfunction. Prior data indicate that mitochondrial dysfunction contributes to hypoglycemia-induced endothelial dysfunction, but the mechanisms behind this linkage remain unknown. We attempt to determine whether clinically relevant low-glucose (LG) exposures acutely induce endothelial dysfunction through activation of the mitochondrial fission process. Characterization of mitochondrial morphology was carried out in cultured endothelial cells by using confocal microscopy. Isolated human arterioles were used to explore the effect LG-induced mitochondrial fission has on the formation of detrimental reactive oxygen species (ROS), bioavailability of nitric oxide (NO), and endothelial-dependent vascular relaxation. Fluorescence microscopy was employed to visualize changes in mitochondrial ROS and NO levels and videomicroscopy applied to measure vasodilation response. Pharmacological disruption of the profission protein Drp1 with Mdivi-1 during LG exposure reduced mitochondrial fragmentation among vascular endothelial cells (LG: 0.469; LG+Mdivi-1: 0.276; P = 0.003), prevented formation of vascular ROS (LG: 2.036; LG+Mdivi-1: 1.774; P = 0.005), increased the presence of NO (LG: 1.352; LG+Mdivi-1: 1.502; P = 0.048), and improved vascular dilation response to acetylcholine (LG: 31.6%; LG+Mdivi-1; 78.5% at maximum dose; P < 0.001). Additionally, decreased expression of Drp1 via siRNA knockdown during LG conditions also improved vascular relaxation. Exposure to LG imparts endothelial dysfunction coupled with altered mitochondrial phenotypes among isolated human arterioles. Disruption of Drp1 and subsequent mitochondrial fragmentation events prevents impaired vascular dilation, restores mitochondrial phenotype, and implicates mitochondrial fission as a primary mediator of LG-induced endothelial dysfunction.NEW & NOTEWORTHY Acute low-glucose exposure induces mitochondrial fragmentation in endothelial cells via Drp1 and is associated with impaired endothelial function in human arterioles. Targeting of Drp1 prevents fragmentation, improves vasofunction, and may provide a therapeutic target for improving cardiovascular complications among diabetics.Listen to this article's corresponding podcast @ http://ajpheart.podbean.com/e/mitochondrial-dynamics-impact-endothelial-function/.


Assuntos
Arteríolas , Endotélio Vascular/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Glucose/deficiência , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/metabolismo , Doenças Vasculares/metabolismo , Adulto , Idoso , Dinaminas , Endotélio Vascular/patologia , Metabolismo Energético/genética , Feminino , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/genética , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Mitocôndrias Cardíacas/patologia , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Óxido Nítrico/metabolismo , Quinazolinonas/farmacologia , RNA Interferente Pequeno/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Doenças Vasculares/patologia , Vasodilatação/efeitos dos fármacos
16.
Vasc Med ; 22(3): 189-196, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28145158

RESUMO

Cell culture and animal work indicate that dipeptidyl peptidase-4 (DPP-4) inhibition may exert cardiovascular benefits through favorable effects on the vascular endothelium. Prior human studies evaluating DPP-4 inhibition have shown conflicting results that may in part be related to heterogeneity of background anti-diabetes therapies. No study has evaluated the acute response of the vasculature to DPP-4 inhibition in humans. We recruited 38 patients with type 2 diabetes on stable background metformin therapy for a randomized, double-blind, placebo-controlled crossover trial of DPP-4 inhibition with sitagliptin (100 mg/day). Each treatment period was 8 weeks long separated by 4 weeks of washout. Endothelial function and plasma markers of endothelial activation (intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)) were measured prior to and 2 hours following acute dosing of sitagliptin or placebo, as well as following 8 weeks of intervention with each pill. Thirty subjects completed the study and were included in analyses. Neither acute nor chronic sitagliptin therapy resulted in significant changes in vascular endothelial function. While post-acute sitagliptin ICAM-1 levels were lower than that post-chronic sitagliptin, the ICAM-1 concentration was not significantly different than pre-acute sitagliptin levels or levels measured in relationship to placebo. There were no significant changes in plasma VCAM-1 levels at any time point. Acute and chronic sitagliptin therapies have neutral effects on the vascular endothelium in the setting of metformin background therapy. In conclusion, our findings suggest DPP-4 inhibition has a neutral effect on cardiovascular risk in patients without a history of heart failure or renal insufficiency. TRIAL REGISTRATION: NCT01859793.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Endotélio Vascular/efeitos dos fármacos , Metformina/uso terapêutico , Fosfato de Sitagliptina/uso terapêutico , Adulto , Idoso , Biomarcadores/sangue , Estudos Cross-Over , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/fisiopatologia , Inibidores da Dipeptidil Peptidase IV/efeitos adversos , Método Duplo-Cego , Quimioterapia Combinada , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Molécula 1 de Adesão Intercelular/sangue , Masculino , Metformina/efeitos adversos , Pessoa de Meia-Idade , Fosfato de Sitagliptina/efeitos adversos , Fatores de Tempo , Resultado do Tratamento , Molécula 1 de Adesão de Célula Vascular/sangue , Vasodilatação/efeitos dos fármacos , Wisconsin , Adulto Jovem
17.
Cardiovasc Diabetol ; 15: 53, 2016 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-27036979

RESUMO

OBJECTIVE: Prior studies demonstrate mitochondrial dysfunction with increased reactive oxygen species generation in peripheral blood mononuclear cells in diabetes mellitus. Oxidative stress-mediated damage to mitochondrial DNA promotes atherosclerosis in animal models. Thus, we evaluated the relation of mitochondrial DNA damage in peripheral blood mononuclear cells s with vascular function in patients with diabetes mellitus and with atherosclerotic cardiovascular disease. APPROACH AND RESULTS: We assessed non-invasive vascular function and mitochondrial DNA damage in 275 patients (age 57 ± 9 years, 60 % women) with atherosclerotic cardiovascular disease alone (N = 55), diabetes mellitus alone (N = 74), combined atherosclerotic cardiovascular disease and diabetes mellitus (N = 48), and controls age >45 without diabetes mellitus or atherosclerotic cardiovascular disease (N = 98). Mitochondrial DNA damage measured by quantitative PCR in peripheral blood mononuclear cells was higher with clinical atherosclerosis alone (0.55 ± 0.65), diabetes mellitus alone (0.65 ± 1.0), and combined clinical atherosclerosis and diabetes mellitus (0.89 ± 1.32) as compared to control subjects (0.23 ± 0.64, P < 0.0001). In multivariable models adjusting for age, sex, and relevant cardiovascular risk factors, clinical atherosclerosis and diabetes mellitus remained associated with higher mitochondrial DNA damage levels (ß = 0.14 ± 0.13, P = 0.04 and ß = 0.21 ± 0.13, P = 0.002, respectively). Higher mitochondrial DNA damage was associated with higher baseline pulse amplitude, a measure of arterial pulsatility, but not with flow-mediated dilation or hyperemic response, measures of vasodilator function. CONCLUSIONS: We found greater mitochondrial DNA damage in patients with diabetes mellitus and clinical atherosclerosis. The association of mitochondrial DNA damage and baseline pulse amplitude may suggest a link between mitochondrial dysfunction and excessive small artery pulsatility with potentially adverse microvascular impact.


Assuntos
Aterosclerose/genética , DNA Mitocondrial/genética , Diabetes Mellitus Tipo 2/genética , Leucócitos Mononucleares/metabolismo , Adulto , Idoso , Aterosclerose/complicações , Aterosclerose/metabolismo , Velocidade do Fluxo Sanguíneo/genética , Artéria Braquial/fisiopatologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Hiperemia/genética , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/genética , Fatores de Risco
18.
Vasc Med ; 20(5): 401-8, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25978968

RESUMO

Prior work suggests blood pressure in African Americans is more sensitive to the effects of aldosterone than in Caucasians. This mechanism may relate to a negative response of the vascular endothelium to aldosterone, including reduced glucose-6-phosphate dehydrogenase (G6PD) activity. Thirty-three African Americans (11 hypertensives, 22 controls) without evidence of diabetes or metabolic syndrome completed the protocol. The protocol included measurement of in vivo microvascular endothelial function by digital pulse arterial tonometry and ex vivo measurement of endothelial function by videomicroscopy of arterioles obtained from these same subjects with and without exposure to aldosterone or spironolactone. Systemic and arteriolar G6PD activities were also measured. In vivo and ex vivo microvascular endothelial function were impaired in African Americans with hypertension. One-hour exposure with aldosterone impaired endothelium-dependent vasodilation in arterioles from normotensive subjects, while 1 hour of spironolactone exposure reversed endothelial dysfunction in arterioles from hypertensive subjects. G6PD activity was impaired in hypertensive arterioles. Aldosterone-related endothelial dysfunction may be responsible for at least a portion of the greater blood pressure sensitivity to aldosterone in African Americans. This may be in part related to vascular suppression of G6PD activity.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Hipertensão/fisiopatologia , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Mineralocorticoides/metabolismo , Espironolactona/metabolismo , Vasodilatação/efeitos dos fármacos , Adulto , Negro ou Afro-Americano , Aldosterona , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Endotélio Vascular/metabolismo , Feminino , Humanos , Masculino
19.
Pediatr Cardiol ; 36(4): 851-61, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25577225

RESUMO

We hypothesized that pediatric patients with type 1 diabetes have cardiac magnetic resonance (CMR) detectable differences in thoracic aortic wall properties and hemodynamics leading to significant local differences in indices of wall shear stress, when compared with age-matched control subjects without diabetes. Pediatric patients with type 1 diabetes were recruited from Children's Hospital of Wisconsin and compared with controls. All underwent morning CMR scanning, 4-limb blood pressure, brachial artery reactivity testing, and venipuncture. Patient-specific computational fluid dynamics modeling with fluid-structure interaction, based on CMR data, determined regional time-averaged wall shear stress (TAWSS) and oscillatory shear index (OSI). Twenty type 1 diabetic subjects, median age 15.8 years (11.6-18.4) and 8 controls 15.4 years (10.3-18.2) were similar except for higher glucose, hemoglobin A1c, and triglycerides for type 1 diabetic subjects. Lower flow-mediated dilation was seen for those with type 1 diabetes (6.5) versus controls (7.8), p = 0.036. For type 1 diabetic subjects, the aorta had more regions with high TAWSS when compared to controls. OSI maps appeared similar. Flow-mediated dilation positively correlated with age at diabetes diagnosis (r = 0.468, p = 0.038) and hemoglobin A1c (r = 0.472, p = 0.036), but did not correlate with aortic distensibility, TAWSS, or OSI. TAWSS did not correlate with any clinical parameter for either group. CMR shows regional differences in aortic wall properties for young diabetic patients. Some local differences in wall shear stress indices were also observed, but a longitudinal study is now warranted.


Assuntos
Sistema Cardiovascular/fisiopatologia , Diabetes Mellitus Tipo 1/complicações , Hemodinâmica , Imagem Cinética por Ressonância Magnética/métodos , Adolescente , Aorta Torácica/patologia , Artéria Braquial/patologia , Estudos de Casos e Controles , Criança , Diabetes Mellitus Tipo 1/patologia , Feminino , Humanos , Masculino , Projetos Piloto , Wisconsin
20.
Am J Physiol Heart Circ Physiol ; 307(1): H25-32, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24778165

RESUMO

Increased intraluminal pressure can reduce endothelial function in resistance arterioles; however, the mechanism of this impairment is unknown. The purpose of this study was to determine the effect of local renin-angiotensin system inhibition on the pressure-induced blunting of endothelium-dependent vasodilation in human adipose arterioles. Arterioles (100-200 µm) were dissected from fresh adipose surgical specimens, cannulated onto glass micropipettes, pressurized to an intraluminal pressure of 60 mmHg, and constricted with endothelin-1. Vasodilation to ACh was assessed at 60 mmHg and again after a 30-min exposure to an intraluminal pressure of 150 mmHg. The vasodilator response to ACh was significantly reduced in vessels exposed to 150 mmHg. Exposure of the vessels to the superoxide scavenger polyethylene glycol-SOD (100 U/ml), the ANG II type 1 receptor antagonist losartan (10(-6) mol/l), or the angiotensin-converting enzyme inhibitor captopril (10(-5) mol/l) prevented the pressure-induced reduction in ACh-dependent vasodilation observed in untreated vessels. High intraluminal pressure had no effect on papaverine-induced vasodilation or ANG II sensitivity. Increased intraluminal pressure increased dihydroethidium fluorescence in cannulated vessels, which could be prevented by polyethylene glycol-SOD or losartan treatment and endothelial denudation. These data indicate that high intraluminal pressure can increase vascular superoxide and reduce nitric oxide-mediated vasodilation via activation of the vascular renin-angiotensin system. This study provides evidence showing that the local renin-angiotensin system in the human microvasculature may be pressure sensitive and contribute to endothelial dysfunction after acute bouts of hypertension.


Assuntos
Tecido Adiposo/fisiologia , Arteríolas/fisiologia , Microcirculação/fisiologia , Óxido Nítrico/metabolismo , Sistema Renina-Angiotensina/fisiologia , Superóxidos/metabolismo , Vasodilatação/fisiologia , Tecido Adiposo/irrigação sanguínea , Velocidade do Fluxo Sanguíneo/fisiologia , Pressão Sanguínea/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
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