RESUMEN
Increasing evidence indicates the role of mitochondrial and vascular dysfunction in aging and aging-associated pathologies; however, the exact mechanisms and chronological processes remain enigmatic. High-energy demand organs, such as the brain, depend on the health of their mitochondria and vasculature for the maintenance of normal functions, therefore representing vulnerable targets for aging. This methodology article describes an analysis pipeline for three-dimensional (3-D) mitochondria-associated signal geometry of two-photon image stacks of brain vasculature. The analysis methods allow the quantification of mitochondria-associated signals obtained in real time in their physiological environment. In addition, signal geometry results will allow the extrapolation of fission and fusion events under normal conditions, during aging, or in the presence of different pathological conditions, therefore contributing to our understanding of the role mitochondria play in a variety of aging-associated diseases with vascular etiology.NEW & NOTEWORTHY Analysis pipeline for 3-D mitochondria-associated signal geometry of two-photon image stacks of brain vasculature.
Asunto(s)
Imagenología Tridimensional , Mitocondrias , Mitocondrias/metabolismo , Animales , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Células Endoteliales/metabolismo , Dinámicas Mitocondriales , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Ratones , Envejecimiento/metabolismoRESUMEN
Mitochondrial numbers and dynamics in brain blood vessels differ between young male and female rats under physiological conditions, but how these differences are affected by stroke is unclear. In males, we found that mitochondrial numbers, possibly due to mitochondrial fission, in large middle cerebral arteries (MCAs) increased following transient middle cerebral artery occlusion (tMCAO). However, mitochondrial effects of stroke on MCAs of female rats have not been studied. To address this disparity, we conducted morphological, biochemical, and functional studies using electron microscopy, Western blot, mitochondrial respiration, and Ca2+ sparks activity measurements in MCAs of female, naïve or sham Sprague-Dawley rats before and 48 h after 90 min of tMCAO. Adverse changes in mitochondrial characteristics and the relationship between mitochondria and sarcoplasmic reticulum (SR) in MCAs were present on both sides. However, mitochondria and mitochondrial/SR associations were often within the range of normal appearance. Mitochondrial protein levels were similar between ipsilateral (ipsi) and contralateral (contra) sides. Nonrespiratory oxygen consumption, maximal respiration, and spare respiratory capacity were similar between ipsi and contra but were reduced compared with sham. Basal respiration, proton leak, and ATP production were similar among MCAs. Ca2+ sparks activity increased in sham and ipsi MCAs exposed to a mitochondrial ATP-sensitive potassium channel opener: diazoxide. Our results show that tMCAO has effects on mitochondria in MCAs on both the ipsi and contra sides. Mitochondrial responses of cerebral arteries to tMCAO in females are substantially different from responses seen previously in male rats suggesting the need for specific sex-based therapies.NEW & NOTEWORTHY We propose that differences in mitochondrial characteristics of males and females, including mitochondrial morphology, respiration, and calcium sparks activity contribute to sex differences in protective and repair mechanisms in response to transient ischemia-reperfusion.
Asunto(s)
Ataque Isquémico Transitorio , Accidente Cerebrovascular , Femenino , Masculino , Ratas , Animales , Arteria Cerebral Media , Ratas Sprague-DawleyRESUMEN
Mitochondria have emerged as a central factor in the pathogenesis and progression of heart failure, and other cardiovascular diseases, as well, but no therapies are available to treat mitochondrial dysfunction. The National Heart, Lung, and Blood Institute convened a group of leading experts in heart failure, cardiovascular diseases, and mitochondria research in August 2018. These experts reviewed the current state of science and identified key gaps and opportunities in basic, translational, and clinical research focusing on the potential of mitochondria-based therapeutic strategies in heart failure. The workshop provided short- and long-term recommendations for moving the field toward clinical strategies for the prevention and treatment of heart failure and cardiovascular diseases by using mitochondria-based approaches.
Asunto(s)
Sistema Cardiovascular , Educación/métodos , Insuficiencia Cardíaca/terapia , Mitocondrias/fisiología , National Heart, Lung, and Blood Institute (U.S.) , Informe de Investigación , Investigación Biomédica/métodos , Investigación Biomédica/tendencias , Sistema Cardiovascular/patología , Educación/tendencias , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/epidemiología , Humanos , National Heart, Lung, and Blood Institute (U.S.)/tendencias , Informe de Investigación/tendencias , Investigación Biomédica Traslacional/métodos , Investigación Biomédica Traslacional/tendencias , Estados Unidos/epidemiologíaRESUMEN
Mitochondria are important regulators of cerebral vascular function in health and disease, but progress in understanding their roles has been hindered by methodological limitations. We report the first in vivo imaging of mitochondria specific to the cerebral endothelium in real time in the same mouse for extended periods. Mice expressing Dendra2 fluorescent protein in mitochondria (mito-Dendra2) in the cerebral vascular endothelium were generated by breeding PhAM-floxed and Tie2-Cre mice. We used mito-Dendra2 expression, cranial window implantation, and two-photon microscopy to visualize mitochondria in the cerebral vascular endothelium of mice. Immunohistochemistry and mitochondrial staining were used to confirm the localization of the mitochondrial signal to endothelial cells and the specificity of mito-Dendra2 to mitochondria. Mito-Dendra2 and Rhodamine B-conjugated dextran allowed simultaneous determinations of mitochondrial density, vessel diameters, area, and mitochondria-to-vessel ratio in vivo, repeatedly, in the same mouse. Endothelial expression of mito-Dendra2 was confirmed in vitro on brain slices and aorta. In addition, we observed an overlapping mito-Dendra2 and Chromeo mitochondrial staining of cultured brain microvascular endothelial cells. Repeated imaging of the same location in the cerebral microcirculation in the same mouse demonstrated stability of mito-Dendra2. While the overall mitochondrial signal was stable over time, mitochondria within the same endothelial cell were mobile. In conclusion, our results indicate that the mito-Dendra2 signal and vascular parameters are suitable for real-time and longitudinal examination of mitochondria in vivo in the cerebral vasculature of mice.NEW & NOTEWORTHY We introduce an innovative in vivo approach to study mitochondria in the cerebral circulation in their physiological environment by demonstrating the feasibility of long-term imaging and three-dimensional reconstruction. We postulate that the appropriate combination of Cre/Lox system and two-photon microscopy will contribute to a better understanding of the role of mitochondria in not only endothelium but also the different cell types of the cerebral circulation.
Asunto(s)
Circulación Cerebrovascular/fisiología , Endotelio Vascular/metabolismo , Mitocondrias/metabolismo , Animales , Células Cultivadas , Células Endoteliales/metabolismo , Femenino , Masculino , Ratones , Ratones Transgénicos , Microscopía de Fluorescencia por Excitación MultifotónicaRESUMEN
One of the major characteristics of hyperglycemic states such as type 2 diabetes is increased reactive oxygen species (ROS) generation. Since mitochondria are a major source of ROS, it is vital to understand the involvement of these organelles in the pathogenesis of ROS-mediated conditions. Therefore, we investigated mitochondrial function and ROS production in cerebral blood vessels of 21-wk-old Zucker diabetic fatty obese rats and their lean controls. We have previously shown that in the early stages of insulin resistance, and short periods of type 2 diabetes mellitus, only mild differences exist in mitochondrial function. In the present study, we examined mitochondrial respiration, mitochondrial protein expression, and ROS production in large-surface cerebral arteries. We used 21-wk-old animals exposed to peak glucose levels for 7 wk and compared them with our previous studies on younger diabetic animals. We found that the same segments of mitochondrial respiration (basal respiration and proton leak) were diminished in diabetic groups as they were in younger diabetic animals. Levels of rattin, a rat humanin analog, tended to decrease in the diabetic group but did not reach statistical significance (P = 0.08). Other mitochondrial proteins were unaffected, which might indicate the existence of compensatory mechanisms with extension of this relatively mild form of diabetes. Superoxide levels were significantly higher in large cerebral vessels of diabetic animals compared with the control group. In conclusion, prolonged dietary diabetes leads to stabilization, rather than deterioration, of metabolic status in the cerebral circulation, despite continued overproduction of ROS.NEW & NOTEWORTHY We have characterized for the first time the dynamics of mitochondrial function during the progression of type 2 diabetes mellitus with regard to mitochondrial respiration, protein expression, and reactive oxygen species production. In addition, this is the first measurement of rattin levels in the cerebral vasculature, which could potentially lead to novel treatment options.
Asunto(s)
Arterias Cerebrales/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Metabolismo Energético , Mitocondrias/metabolismo , Animales , Glucemia/metabolismo , Respiración de la Célula , Arterias Cerebrales/patología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Masculino , Mitocondrias/patología , Proteínas/metabolismo , Ratas Zucker , Superóxidos/metabolismo , Factores de TiempoRESUMEN
Mitochondrial dysfunction has been suggested as a potential underlying cause of pathological conditions associated with type 2 diabetes (T2DM). We have previously shown that mitochondrial respiration and mitochondrial protein levels were similar in the large cerebral arteries of insulin-resistant Zucker obese rats and their lean controls. In this study, we extend our investigations into the mitochondrial dynamics of the cerebral vasculature of 14-week-old Zucker diabetic fatty obese (ZDFO) rats with early T2DM. Body weight and blood glucose levels were significantly higher in the ZDFO group, and basal mitochondrial respiration and proton leak were significantly decreased in the large cerebral arteries of the ZDFO rats compared with the lean controls (ZDFL). The expression of the mitochondrial proteins total manganese superoxide dismutase (MnSOD) and voltage-dependent anion channel (VDAC) were significantly lower in the cerebral microvessels, and acetylated MnSOD levels were significantly reduced in the large arteries of the ZDFO group. Additionally, superoxide production was significantly increased in the microvessels of the ZDFO group. Despite evidence of increased oxidative stress in ZDFO, exogenous SOD was not able to restore mitochondrial respiration in the ZDFO rats. Our results show, for the first time, that mitochondrial respiration and protein levels are compromised during the early stages of T2DM.
Asunto(s)
Arterias Cerebrales/metabolismo , Trastornos Cerebrovasculares/etiología , Diabetes Mellitus Tipo 2/complicaciones , Angiopatías Diabéticas/etiología , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Acetilación , Animales , Glucemia/metabolismo , Peso Corporal , Respiración de la Célula , Arterias Cerebrales/efectos de los fármacos , Trastornos Cerebrovasculares/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Angiopatías Diabéticas/metabolismo , Modelos Animales de Enfermedad , Depuradores de Radicales Libres/farmacología , Masculino , Microvasos/metabolismo , Mitocondrias/efectos de los fármacos , Dinámicas Mitocondriales/efectos de los fármacos , Estrés Oxidativo , Ratas Zucker , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , Factores de Tiempo , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
Little is known about mitochondrial functioning in the cerebral vasculature during insulin resistance (IR). We examined mitochondrial respiration in isolated cerebral arteries of male Zucker obese (ZO) rats and phenotypically normal Zucker lean (ZL) rats using the Seahorse XFe24 analyzer. We investigated mitochondrial morphology in cerebral blood vessels as well as mitochondrial and nonmitochondrial protein expression levels in cerebral arteries and microvessels. We also measured reactive oxygen species (ROS) levels in cerebral microvessels. Under basal conditions, the mitochondrial respiration components (nonmitochondrial respiration, basal respiration, ATP production, proton leak, and spare respiratory capacity) showed similar levels among the ZL and ZO groups with the exception of maximal respiration, which was higher in the ZO group. We examined the role of nitric oxide by measuring mitochondrial respiration following inhibition of nitric oxide synthase with N(ω)-nitro-l-arginine methyl ester (l-NAME) and mitochondrial activation after administration of diazoxide (DZ). Both ZL and ZO groups showed similar responses to these stimuli with minor variations.l-NAME significantly increased the proton leak, and DZ decreased nonmitochondrial respiration in the ZL group. Other components were not affected. Mitochondrial morphology and distribution within vascular smooth muscle and endothelium as well as mitochondrial protein levels were similar in the arteries and microvessels of both groups. Endothelial nitric oxide synthase (eNOS) and ROS levels were increased in cerebral microvessels of the ZO. Our study suggests that mitochondrial function is not significantly altered in the cerebral vasculature of young ZO rats, but increased ROS production might be due to increased eNOS in the cerebral microcirculation during IR.
Asunto(s)
Arterias Cerebrales/metabolismo , Resistencia a la Insulina , Microvasos/metabolismo , Mitocondrias/metabolismo , Obesidad/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Respiración de la Célula , Endotelio Vascular/metabolismo , Masculino , Músculo Liso Vascular/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ratas , Ratas Zucker , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The diverse signaling events following mitochondrial depolarization in neurons are not clear. We examined for the first time the effects of mitochondrial depolarization on mitochondrial function, intracellular calcium, neuronal nitric oxide synthase (nNOS) activation, and nitric oxide (NO) production in cultured neurons and perivascular nerves. Cultured rat primary cortical neurons were studied on 7-10 days in vitro, and endothelium-denuded cerebral arteries of adult Sprague-Dawley rats were studied ex vivo. Diazoxide and BMS-191095 (BMS), activators of mitochondrial KATP channels, depolarized mitochondria in cultured neurons and increased cytosolic calcium levels. However, the mitochondrial oxygen consumption rate was unaffected by mitochondrial depolarization. In addition, diazoxide and BMS not only increased the nNOS phosphorylation at positive regulatory serine 1417 but also decreased nNOS phosphorylation at negative regulatory serine 847. Furthermore, diazoxide and BMS increased NO production in cultured neurons measured with both fluorescence microscopy and electron spin resonance spectroscopy, which was sensitive to inhibition by the selective nNOS inhibitor 7-nitroindazole (7-NI). Diazoxide also protected cultured neurons against oxygen-glucose deprivation, which was blocked by NOS inhibition and rescued by NO donors. Finally, BMS induced vasodilation of endothelium denuded, freshly isolated cerebral arteries that was diminished by 7-NI and tetrodotoxin. Thus pharmacological depolarization of mitochondria promotes activation of nNOS leading to generation of NO in cultured neurons and endothelium-denuded arteries. Mitochondrial-induced NO production leads to increased cellular resistance to lethal stress by cultured neurons and to vasodilation of denuded cerebral arteries.
Asunto(s)
Arterias Cerebrales/metabolismo , Potencial de la Membrana Mitocondrial , Mitocondrias/enzimología , Neuronas Nitrérgicas/enzimología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico/metabolismo , Comunicación Paracrina , Vasodilatación , Animales , Benzopiranos/farmacología , Células Cultivadas , Arterias Cerebrales/efectos de los fármacos , Arterias Cerebrales/inervación , Diazóxido/farmacología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Indazoles/farmacología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Neuronas Nitrérgicas/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Comunicación Paracrina/efectos de los fármacos , Fosforilación , Canales de Potasio/agonistas , Canales de Potasio/metabolismo , Cultivo Primario de Células , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Serina , Transducción de Señal , Vasodilatación/efectos de los fármacosRESUMEN
We examined the role of the mechanistic target of rapamycin (mTOR) pathway in delayed diazoxide (DZ)-induced preconditioning of cultured rat primary cortical neurons. Neurons were treated for 3 days with 500 µM DZ or feeding medium and then exposed to 3 h of continuous normoxia in Dulbecco's modified eagle medium with glucose or with 3 h of oxygen-glucose deprivation (OGD) followed by normoxia and feeding medium. The OGD decreased viability by 50%, depolarized mitochondria, and reduced mitochondrial respiration, whereas DZ treatment improved viability and mitochondrial respiration, and suppressed reactive oxygen species production, but did not restore mitochondrial membrane potential after OGD. Neuroprotection by DZ was associated with increased phosphorylation of protein kinase B (Akt), mTOR, and the major mTOR downstream substrate, S6 Kinase (S6K). The mTOR inhibitors rapamycin and Torin-1, as well as S6K-targeted siRNA abolished the protective effects of DZ. The effects of DZ on mitochondrial membrane potential and reactive oxygen species production were not affected by rapamycin. Preconditioning with DZ also changed mitochondrial and non-mitochondrial oxygen consumption rates. We conclude that in addition to reducing reactive oxygen species (ROS) production and mitochondrial membrane depolarization, DZ protects against OGD by activation of the Akt-mTOR-S6K pathway and by changes in mitochondrial respiration. Ischemic strokes have limited therapeutic options. Diazoxide (DZ) preconditioning can reduce neuronal damage. Using oxygen-glucose deprivation (OGD), we studied Akt/mTOR/S6K signaling and mitochondrial respiration in neuronal preconditioning. We found DZ protects neurons against OGD via the Akt/mTOR/S6K pathway and alters the mitochondrial and non-mitochondrial oxygen consumption rate. This suggests that the Akt/mTOR/S6k pathway and mitochondria are novel stroke targets.
Asunto(s)
Diazóxido/farmacología , Precondicionamiento Isquémico , Proteínas del Tejido Nervioso/fisiología , Neuronas/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas/fisiología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/fisiología , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/embriología , Medios de Cultivo/farmacología , Activación Enzimática/efectos de los fármacos , Técnicas In Vitro , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Neuronas/metabolismo , Oxígeno/farmacología , Consumo de Oxígeno , Fosforilación , Cultivo Primario de Células , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas/genética , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
Mitochondrial respiration has never been directly examined in intact cerebral arteries. We tested the hypothesis that mitochondrial energetics of large cerebral arteries ex vivo are sex dependent. The Seahorse XFe24 analyzer was used to examine mitochondrial respiration in isolated cerebral arteries from adult male and female Sprague-Dawley rats. We examined the role of nitric oxide (NO) on mitochondrial respiration under basal conditions, using N(ω)-nitro-l-arginine methyl ester, and following pharmacological challenge using diazoxide (DZ), and also determined levels of mitochondrial and nonmitochondrial proteins using Western blot, and vascular diameter responses to DZ. The components of mitochondrial respiration including basal respiration, ATP production, proton leak, maximal respiration, and spare respiratory capacity were elevated in females compared with males, but increased in both male and female arteries in the presence of the NOS inhibitor. Although acute DZ treatment had little effect on mitochondrial respiration of male arteries, it decreased the respiration in female arteries. Levels of mitochondrial proteins in Complexes I-V and the voltage-dependent anion channel protein were elevated in female compared with male cerebral arteries. The DZ-induced vasodilation was greater in females than in males. Our findings show that substantial sex differences in mitochondrial respiratory dynamics exist in large cerebral arteries and may provide the mechanistic basis for observations that the female cerebral vasculature is more adaptable after injury.
Asunto(s)
Arterias Cerebrales/efectos de los fármacos , Diazóxido/farmacología , Factores Relajantes Endotelio-Dependientes/farmacología , Inhibidores Enzimáticos/farmacología , Mitocondrias/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/farmacología , Vasodilatadores/farmacología , Adenosina Trifosfato/metabolismo , Animales , Respiración de la Célula/efectos de los fármacos , Arterias Cerebrales/metabolismo , Femenino , Masculino , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Factores SexualesRESUMEN
The objective of the present study was to determine whether mitochondrial function in the cerebral vasculature is maintained after transient middle cerebral artery (MCA) occlusion (tMCAO) in rats. Sprague-Dawley rats were exposed to 90 min of tMCAO followed by 4 or 48 h of reperfusion. MCAs from ischemic (ipsilateral) and nonischemic (contralateral) sides were compared with control MCAs from sham-operated rats. We determined 1) vasoreactivity to diazoxide (DZ; a mitochondrial ATP-activated K(+) channel opener), ACh, bradykinin (BK), serotonin, and sodium nitroprusside; 2) levels of mitochondrial and nonmitochondrial proteins and mitochondrial DNA; and 3) vascular levels of tetramethylrhodamine ethyl ester (an indicator of mitochondrial membrane potential). All dilator responses, including those with DZ, were intact 4 h post-tMCAO. Dilator responses to ACh, BK, and sodium nitroprusside were reduced in ipsilateral MCAs at 48 h compared with contralateral MCAs, but DZ responses were comparable with control MCAs. Surprisingly, contralateral responses to ACh, BK, and serotonin were reduced compared with control MCAs at 48 h. Ipsilateral vasodilation to DZ at 48 h was eliminated by endothelial denudation and endothelial nitric oxide synthase (eNOS) inhibition but was only reduced in control MCAs. Mitochondrial proteins, phosphorylated eNOS, mitochondrial DNA, and mitochondrial membrane potential were higher in ipsilateral compared with contralateral MCAs. In conclusion, contrary to conventional wisdom, mitochondria remain functional for at least 48 h after severe ischemic stress in MCAs, and DZ-induced dilation is preserved due to maintained mitochondrial mass, probably in the endothelium, and eNOS signaling. Our findings support the concept that functioning vascular mitochondria are an unexpected target for novel stroke therapies.
Asunto(s)
Arterias Cerebrales/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Mitocondrias/metabolismo , Acetilcolina/farmacología , Animales , Bradiquinina/farmacología , Arterias Cerebrales/efectos de los fármacos , Arterias Cerebrales/fisiología , Diazóxido/farmacología , Masculino , Potencial de la Membrana Mitocondrial , Mitocondrias/fisiología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitroprusiato/farmacología , Ratas , Ratas Sprague-Dawley , Serotonina/farmacología , VasoconstricciónRESUMEN
Mitochondrial depolarization following ATP-sensitive potassium (mitoKATP) channel activation has been shown to induce cerebral vasodilation by generation of mitochondrial reactive oxygen species (ROS), which sequentially promotes frequency of calcium sparks and activation of large conductance calcium-activated potassium channels (BKCa) in vascular smooth muscle (VSM). We previously demonstrated that cerebrovascular insulin resistance accompanies aging and obesity. It is unclear whether mitochondrial depolarization without the ROS generation enhances calcium sparks and vasodilation in phenotypically normal [Sprague Dawley (SD); Zucker lean (ZL)] and insulin-resistant [Zucker obese (ZO)] rats. We compared the mechanisms underlying the vasodilation to ROS-dependent (diazoxide) and ROS-independent [BMS-191095 (BMS)] mitoKATP channel activators in normal and ZO rats. Arterial diameter studies from SD, ZL, and ZO rats showed that BMS as well as diazoxide induced vasodilation in endothelium-denuded cerebral arteries. In normal rats, BMS-induced vasodilation was mediated by mitochondrial depolarization and calcium sparks generation in VSM and was reduced by inhibition of BKCa channels. However, unlike diazoxide-induced vasodilation, scavenging of ROS had no effect on BMS-induced vasodilation. Electron spin resonance spectroscopy confirmed that diazoxide but not BMS promoted vascular ROS generation. BMS- as well as diazoxide-induced vasodilation, mitochondrial depolarization, and calcium spark generation were diminished in cerebral arteries from ZO rats. Thus pharmacological depolarization of VSM mitochondria by BMS promotes ROS-independent vasodilation via generation of calcium sparks and activation of BKCa channels. Diminished generation of calcium sparks and reduced vasodilation in ZO arteries in response to BMS and diazoxide provide new insights into mechanisms of cerebrovascular dysfunction in insulin resistance.
Asunto(s)
Arterias Cerebrales/metabolismo , Resistencia a la Insulina , Mitocondrias Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Vasodilatación , Animales , Benzopiranos/farmacología , Señalización del Calcio , Arterias Cerebrales/fisiología , Diazóxido/farmacología , Imidazoles/farmacología , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Potencial de la Membrana Mitocondrial , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Canales de Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Zucker , Especies Reactivas de Oxígeno/metabolismo , Vasodilatadores/farmacologíaRESUMEN
Mitochondrial-initiated events protect the neurovascular unit against lethal stress via a process called preconditioning, which independently promotes changes in cerebrovascular tone through shared signaling pathways. Activation of adenosine triphosphate (ATP)-dependent potassium channels on the inner mitochondrial membrane (mitoKATP channels) is a specific and dependable way to induce protection of neurons, astroglia, and cerebral vascular endothelium. Through the opening of mitoKATP channels, mitochondrial depolarization leads to activation of protein kinases and transient increases in cytosolic calcium (Ca(2+)) levels that activate terminal mechanisms that protect the neurovascular unit against lethal stress. The release of reactive oxygen species from mitochondria has similar protective effects. Signaling elements of the preconditioning pathways also are involved in the regulation of vascular tone. Activation of mitoKATP channels in cerebral arteries causes vasodilation, with cell-specific contributions from the endothelium, vascular smooth muscles, and nerves. Preexisting chronic conditions, such as insulin resistance and/or diabetes, prevent preconditioning and impair relaxation to mitochondrial-centered responses in cerebral arteries. Surprisingly, mitochondrial activation after anoxic or ischemic stress appears to protect cerebral vascular endothelium and promotes the restoration of blood flow; therefore, mitochondria may represent an important, but underutilized target in attenuating vascular dysfunction and brain injury in stroke patients.
Asunto(s)
Encéfalo/irrigación sanguínea , Mitocondrias/fisiología , Canales de Potasio/fisiología , Bencimidazoles/farmacología , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/fisiopatología , Arterias Cerebrales , Precondicionamiento Isquémico , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Músculo Liso Vascular/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
OBJECTIVE: Mitochondrial depolarization after ATP-sensitive potassium channel activation has been shown to induce cerebral vasodilation by the generation of calcium sparks in smooth muscle. It is unclear, however, whether mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitochondrial ATP-sensitive potassium channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation. APPROACH AND RESULTS: Diameter studies in isolated rat cerebral arteries showed BMS- and diazoxide-induced vasodilations that were diminished by endothelial denudation. Mitochondrial depolarization-induced vasodilation was reduced by inhibition of mitochondrial ATP-sensitive potassium channels, phosphoinositide-3 kinase, or nitric oxide synthase. Scavenging of reactive oxygen species, however, diminished vasodilation induced by diazoxide, but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells showed that BMS elicited mitochondrial depolarization and enhanced nitric oxide production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial reactive oxygen species production. Measurements of intracellular calcium ([Ca(2+)]i) in cultured rat brain microvascular endothelial cells and arteries showed that both diazoxide and BMS increased endothelial [Ca(2+)]i. Western blot analyses revealed increased phosphorylation of protein kinase B and endothelial nitric oxide synthase (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by phosphoinositide-3 kinase inhibition. Electron spin resonance spectroscopy confirmed vascular nitric oxide generation in response to diazoxide and BMS. CONCLUSIONS: Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca(2+)]i as well as by phosphoinositide-3 kinase-protein kinase B-induced eNOS phosphorylation. Both mitochondrial reactive oxygen species-dependent and -independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.
Asunto(s)
Arterias Cerebrales/metabolismo , Circulación Cerebrovascular , Células Endoteliales/metabolismo , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Canales de Potasio/metabolismo , Vasodilatación , Animales , Benzopiranos/farmacología , Western Blotting , Calcio/metabolismo , Células Cultivadas , Arterias Cerebrales/efectos de los fármacos , Circulación Cerebrovascular/efectos de los fármacos , Diazóxido/farmacología , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Células Endoteliales/efectos de los fármacos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Imidazoles/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/agonistas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
We previously reported evidence that oxidative stress during aging leads to adverse protein profile changes of brain cortical microvessels (MVs: end arterioles, capillaries, and venules) that affect mRNA/protein stability, basement membrane integrity, and ATP synthesis capacity in mice. As an extension of our previous study, we also found that proteins which comprise the blood-brain barrier (BBB) and regulate mitochondrial quality control were also significantly decreased in the mice's cortical MVs with aging. Interestingly, the neuroinflammatory protein fibrinogen (Fgn) was increased in mice brain MVs, which corresponds with clinical reports indicating that the plasma Fgn concentration increased progressively with aging. In this study, protein-protein interaction network analysis indicated that high expression of Fgn is linked with downregulated expression of both BBB- and mitochondrial fission/fusion-related proteins in mice cortical MVs with aging. To investigate the mechanism of Fgn action, we observed that 2 mg/mL or higher concentration of human plasma Fgn changed cell morphology, induced cytotoxicity, and increased BBB permeability in primary human brain microvascular endothelial cells (HBMECs). The BBB tight junction proteins were significantly decreased with increasing concentration of human plasma Fgn in primary HBMECs. Similarly, the expression of phosphorylated dynamin-related protein 1 (pDRP1) and other mitochondrial fission/fusion-related proteins were also significantly reduced in Fgn-treated HBMECs. Interestingly, DRP1 knockdown by shRNA(h) resulted in the reduction of both BBB- and mitochondrial fission/fusion-related proteins in HBMECs. Our results suggest that elevated Fgn downregulates DRP1, leading to mitochondrial-dependent endothelial and BBB dysfunction in the brain microvasculature.
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Barrera Hematoencefálica , Células Endoteliales , Ratones , Humanos , Animales , Barrera Hematoencefálica/metabolismo , Fibrinógeno/metabolismo , Microvasos/metabolismo , Dinaminas/metabolismoRESUMEN
Cognitive impairment and dementias during aging such as Alzheimer's disease are linked to functional decline and structural alterations of the brain microvasculature. Although mechanisms leading to microvascular changes during aging are not clear, loss of mitochondria, and reduced efficiency of remaining mitochondria appear to play a major role. Pharmacological agents, such as SS-31, which target mitochondria have been shown to be effective during aging and diseases; however, the benefit to mitochondrial- and non-mitochondrial proteins in the brain microvasculature has not been examined. We tested whether attenuation of aging-associated changes in the brain microvascular proteome via targeting mitochondria represents a therapeutic option for the aging brain. We used aged male (> 18 months) C57Bl6/J mice treated with a mitochondria-targeted tetrapeptide, SS-31, or vehicle saline. Cerebral blood flow (CBF) was determined using laser speckle imaging during a 2-week treatment period. Then, isolated cortical microvessels (MVs) composed of end arterioles, capillaries, and venules were used for Orbitrap Eclipse Tribrid mass spectrometry. CBF was similar among the groups, whereas bioinformatic analysis revealed substantial differences in protein abundance of cortical MVs between SS-31 and vehicle. We identified 6267 proteins, of which 12% were mitochondria-associated. Of this 12%, 107 were significantly differentially expressed and were associated with oxidative phosphorylation, metabolism, the antioxidant defense system, or mitochondrial dynamics. Administration of SS-31 affected many non-mitochondrial proteins. Our findings suggest that mitochondria in the microvasculature represent a therapeutic target in the aging brain, and widespread changes in the proteome may underlie the rejuvenating actions of SS-31 in aging.
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Proteoma , Proteómica , Ratones , Animales , Masculino , Proteoma/metabolismo , Proteoma/farmacología , Proteómica/métodos , Mitocondrias/metabolismo , Encéfalo/metabolismo , Microvasos/metabolismoRESUMEN
Despite the suppression of human immunodeficiency virus (HIV) replication by combined antiretroviral therapy (cART), 50-60% of HIV-infected patients suffer from HIV-associated neurocognitive disorders (HAND). Studies are uncovering the role of extracellular vesicles (EVs), especially exosomes, in the central nervous system (CNS) due to HIV infection. We investigated links among circulating plasma exosomal (crExo) proteins and neuropathogenesis in simian/human immunodeficiency virus (SHIV)-infected rhesus macaques (RM) and HIV-infected and cART treated patients (Patient-Exo). Isolated EVs from SHIV-infected (SHIV-Exo) and uninfected (CTL-Exo) RM were predominantly exosomes (particle size < 150 nm). Proteomic analysis quantified 5654 proteins, of which 236 proteins (~4%) were significantly, differentially expressed (DE) between SHIV-/CTL-Exo. Interestingly, different CNS cell specific markers were abundantly expressed in crExo. Proteins involved in latent viral reactivation, neuroinflammation, neuropathology-associated interactive as well as signaling molecules were expressed at significantly higher levels in SHIV-Exo than CTL-Exo. However, proteins involved in mitochondrial biogenesis, ATP production, autophagy, endocytosis, exocytosis, and cytoskeleton organization were significantly less expressed in SHIV-Exo than CTL-Exo. Interestingly, proteins involved in oxidative stress, mitochondrial biogenesis, ATP production, and autophagy were significantly downregulated in primary human brain microvascular endothelial cells exposed with HIV+/cART+ Patient-Exo. We showed that Patient-Exo significantly increased blood-brain barrier permeability, possibly due to loss of platelet endothelial cell adhesion molecule-1 protein and actin cytoskeleton structure. Our novel findings suggest that circulating exosomal proteins expressed CNS cell markers-possibly associated with viral reactivation and neuropathogenesis-that may elucidate the etiology of HAND.
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Infecciones por VIH , VIH-1 , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios , Animales , Humanos , Macaca mulatta , Infecciones por VIH/complicaciones , Síndrome de Inmunodeficiencia Adquirida del Simio/complicaciones , Células Endoteliales , Proteómica , Modelos Animales de Enfermedad , Adenosina Trifosfato , Carga ViralRESUMEN
OBJECTIVE: Previously, we have shown that IR impairs the vascular reactivity of the major cerebral arteries of ZO rats prior to the occurrence of Type-II diabetes mellitus. However, the functional state of the microcirculation in the cerebral cortex is still being explored. METHODS: We tested the local CoBF responses of 11-13-week-old ZO (n = 31) and control ZL (n = 32) rats to several stimuli measured by LDF using a closed cranial window setup. RESULTS: The topical application of 1-100 µm bradykinin elicited the same degree of CoBF elevation in both ZL and ZO groups. There was no significant difference in the incidence, latency, and amplitude of the NMDA-induced CSD-related hyperemia between the ZO and ZL groups. Hypercapnic CoBF response to 5% carbon-dioxide ventilation did not significantly change in the ZO compared with the ZL. Topical bicuculline-induced cortical seizure was accompanied by the same increase of CoBF in both the ZO and ZL at all bicuculline doses. CONCLUSIONS: CoBF responses of the microcirculation are preserved in the early period of the metabolic syndrome, which creates an opportunity for intervention to prevent and restore the function of the major cerebral vascular beds.
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Bicuculina/farmacología , Circulación Cerebrovascular/efectos de los fármacos , Convulsivantes/farmacología , Diabetes Mellitus Tipo 2/fisiopatología , Resistencia a la Insulina , Microcirculación/efectos de los fármacos , Animales , Bicuculina/efectos adversos , Convulsivantes/efectos adversos , Diabetes Mellitus Tipo 2/metabolismo , Hipercapnia/metabolismo , Hipercapnia/fisiopatología , Masculino , Ratas , Convulsiones/inducido químicamente , Convulsiones/metabolismo , Convulsiones/fisiopatologíaRESUMEN
ANG II-stimulated production of reactive oxygen species (ROS) through NADPH oxidase is suggested to activate MAPK pathways, which are implicated in neurally mediated pressor effects of ANG II. Emerging evidence suggests that ANG-(1-7) up regulates MAPK phosphatases to reduce MAPK signaling and attenuate actions of ANG II. Whether angiotensin peptides participate in long-term regulation of these systems in the brain is not known. Therefore, we determined tissue and mitochondrial ROS, as well as expression and activity of MAPK phosphatase-1 (MKP-1) in brain dorsal medullary tissue of hypertensive transgenic (mRen2)27 rats exhibiting higher ANG II/ANG-(1-7) tone or hypotensive transgenic rats with targeted decreased glial expression of angiotensinogen, ASrAOGEN (AS) exhibiting lower ANG II/ANG-(1-7) tone compared with normotensive Sprague-Dawley (SD) rats that serve as the control strain. Transgenic (mRen2)27 rats showed higher medullary tissue NADPH oxidase activity and dihydroethidium fluorescence in isolated mitochondria vs. SD or AS rats. Mitochondrial uncoupling protein 2 was lower in AS and unchanged in (mRen2)27 compared with SD rats. MKP-1 mRNA and protein expression were higher in AS and unchanged in (mRen2)27 compared with SD rats. AS rats also had lower phosphorylated ERK1/2 and JNK consistent with higher MKP-1 activity. Thus, an altered brain renin-angiotensin system influences oxidative stress status and regulates MKP-1 expression. However, there is a dissociation between these effects and the hemodynamic profiles. Higher ROS was associated with hypertension in (mRen2)27 and normal MKP-1, whereas the higher MKP-1 was associated with hypotension in AS, where ROS was normal relative to SD rats.
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Fosfatasa 1 de Especificidad Dual/metabolismo , Hipertensión/enzimología , Bulbo Raquídeo/enzimología , Estrés Oxidativo , Sistema Renina-Angiotensina , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Angiotensinógeno/genética , Angiotensinógeno/metabolismo , Animales , Presión Sanguínea , Modelos Animales de Enfermedad , Fosfatasa 1 de Especificidad Dual/genética , Regulación de la Expresión Génica , Hipertensión/genética , Hipertensión/fisiopatología , Canales Iónicos/genética , Canales Iónicos/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Mitocondrias/enzimología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , NADPH Oxidasas/metabolismo , Oligonucleótidos Antisentido/metabolismo , Fragmentos de Péptidos/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Especies Reactivas de Oxígeno/metabolismo , Renina/genética , Renina/metabolismo , Sistema Renina-Angiotensina/genética , Transducción de Señal , Proteína Desacopladora 2RESUMEN
Differentially expressed (DE) proteins in the cortical microvessels (MVs) of young, middle-aged, and old male and female mice were evaluated using discovery-based proteomics analysis (> 4,200 quantified proteins/group). Most DE proteins (> 90%) showed no significant differences between the sexes; however, some significant DE proteins showing sexual differences in MVs decreased from young (8.3%), to middle-aged (3.7%), to old (0.5%) mice. Therefore, we combined male and female data for age-dependent comparisons but noted sex differences for examination. Key proteins involved in the oxidative stress response, mRNA or protein stability, basement membrane (BM) composition, aerobic glycolysis, and mitochondrial function were significantly altered with aging. Relative abundance of superoxide dismutase-1/-2, catalase and thioredoxin were reduced with aging. Proteins participating in either mRNA degradation or pre-mRNA splicing were significantly increased in old mice MVs, whereas protein stabilizing proteins decreased. Glycolytic proteins were not affected in middle age, but the relative abundance of these proteins decreased in MVs of old mice. Although most of the 41 examined proteins composing mitochondrial complexes I-V were reduced in old mice, six of these proteins showed a significant reduction in middle-aged mice, but the relative abundance increased in fourteen proteins. Nidogen, collagen, and laminin family members as well as perlecan showed differing patterns during aging, indicating BM reorganization starting in middle age. We suggest that increased oxidative stress during aging leads to adverse protein profile changes of brain cortical MVs that affect mRNA/protein stability, BM integrity, and ATP synthesis capacity.