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Cellular senescence plays a pivotal role in the aging and progression of neurodegenerative diseases, including vascular cognitive impairment and dementia (VCID). In postmortem brains from individuals with VCID, endothelin-1 (ET-1) levels closely correlate with blood barrier breakdown and cerebral hypoperfusion. Brain microvascular endothelial cells (BMVECs), previously thought to have exclusively endothelin B receptors, also possess endothelin A (ETA) receptors; however, the functional significance of this receptor in BMVECs is not known. We hypothesize that ETA receptors mediate BMVEC senescence. Serumstarved human BMVECs (HBEC5i) were incubated with ET-1 (1 [micro]mol/L) in the presence/absence of ETA receptor antagonist BQ-123 (20 [micro]mol/L). Cells were collected for Western blot and quantitative real-time PCR analyses. Treatment of ET-1 increased protein expression of ETA receptor, while it was prevented by the ETA receptor antagonist. ET-1 increased p21, p16, p53, LIF1 and cyclin D1 protein levels, and [beta]-galactosidase accumulation, which were prevented in the presence of ETA blockade. While there was no change in tight junction proteins, ET-1 decreased adherent junction protein vascular endothelial cadherin (VEcadherin) levels. In conclusion, ET-1 upregulates ETA receptors in BMVECs in an autocrine manner and triggers the activation of senescence. These in vitro findings need to be further studied in vivo to establish the role of ETA receptors in the progression of endothelial senescence in VCID. Key words: endothelial cells, endothelin-1, senescence, brain La senescence cellulaire joue un role central dans le vieillissement et l'evolution des maladies neurodegeneratives, y compris les deficits cognitifs et la demence vasculaires (DCDV). Dans le cerveau autopsie de personnes atteintes de DCDV, les taux d'endotheline 1 (ET-1) sont etroitement proportionnels a l'effacement de la barriere hemoencephalique et a l'hypoperfusion cerebrale. Les cellules endotheliales des microvaisseaux du cerveau (CEMVC) que l'on croyait precedemment exprimer uniquement des recepteurs ETB, possedent aussi des recepteurs ETA, mais on ne connait pas le role fonctionnel de ces recepteurs dans les CEMVC. Nous avons formule une hypothese selon laquelle les recepteurs ETA participent a la mediation de la senescence des CEMVC. Nous avons incube des cellules humaines CEMVC carencee en serum (HBEC5i) avec de l'ET-1 (a 1 [micro]M), en presence et en absence de BQ123 (a 20 [micro]M), un antagoniste des recepteurs ETA. Nous avons recueilli les cellules en vue d'analyses par immunobuvardage de Western et de PCR quantitatif en temps reel. L'administration d'ET-1 a entraine une augmentation de l'expression proteique des recepteurs ETA, laquelle etait inhibee avec l'antagoniste des recepteur ETA. L'ET-1 a entraine une hausse de p21, de p16 et de p53, de meme que des taux de LIF1 et de cycline D1 en proteines, ainsi que l'accumulation de [beta]-galactosidase, ce qui etait inhibe avec le blocage des recepteurs ETA. Bien qu'aucune modification des proteines des jonctions serrees n'ait ete observee, l'ET-1 a entraine une diminution des taux de VE-cadherine, proteine de jonction adherente. En conclusion, l'ET-1 entraine une regulation a la hausse des recepteurs ETA dans les CEMVC par un mode autocrine, de meme que le declenchement de l'activation de la senescence. Il est necessaire que ces resultats in vitro soient etudies davantage in vivo en vue d'etablir le role des recepteurs ETA dans l'evolution de la senescence de l'endothelium dans les DCDV. Mots-cles : cellules endotheliales, endotheline 1, senescence, cerveau, Introduction Brain health depends upon cardiovascular health. Endothelial dysfunction leads to improper regulation of cerebral blood flow (CBF) and disruption of blood-brain barrier (BBB) integrity. As such, it is not [...]

Endothelin-1 (ET-1), the most potent vasoconstrictor identified to date, contributes to cerebrovascular dysfunction. ET-1 levels in postmortem brain specimens from individuals diagnosed with Alzheimer's disease (AD) and related dementias (ADRD) were shown to be related to cerebral hypoxia and disease severity. ET-1-mediated vascular dysfunction and ensuing cognitive deficits have also been reported in experimental models of AD and ADRD. Moreover, studies also showed that ET-1 secreted from brain microvascular endothelial cells (BMVECs) can affect neurovascular unit integrity in an autocrine and paracrine manner. Vascular contributions to cognitive impairment and dementia (VCID) is a leading ADRD cause known to be free of neuronal tau pathology, a hallmark of AD. However, a recent study reported cytotoxic hyperphosphorylated tau (p-tau) accumulation, which fails to bind or stabilize microtubules in BMVECs in VCID. Thus, the study aimed to determine the impact of ET-1 on tau pathology, microtubule organization, and barrier function in BMVECs. Cells were stimulated with 1 μM ET-1 for 24 h in the presence/absence of ETA (BQ123; 20 μM) or ETB (BQ788; 20 μM) receptor antagonists. Cell lysates were assayed for an array of phosphorylation site-specific antibodies and microtubule organization/stabilization markers. ET-1 stimulation increased p-tau Thr231 but decreased p-tau Ser199, Ser262, Ser396, and Ser214 levels only in the presence of ETA or ETB antagonism. ET-1 also impaired barrier function in the presence of ETA antagonism. These novel findings suggest that (1) dysregulation of endothelial tau phosphorylation may contribute to cerebral microvascular dysfunction and (2) the ET system may be an early intervention target to prevent hyperphosphorylated tau-mediated disruption of BMVEC barrier function. [ABSTRACT FROM AUTHOR]

Diabetes mediates endothelial dysfunction and increases the risk of Alzheimer's disease and related dementias. Diabetes also dysregulates the ET system. ET-1-mediated constriction of brain microvascular pericytes (BMVPCs) has been shown to contribute to brain hypoperfusion. Cellular senescence, a process that arrests the proliferation of harmful cells and instigates phenotypical changes and proinflammatory responses in endothelial cells that impact their survival and function. Thus, we hypothesized that ET-1 mediates BMVPC senescence and phenotypical changes in diabetes-like conditions. Human BMVPCs were incubated in diabetes-like conditions with or without ET-1 (1 µmol/L) for 3 and 7 days. Hydrogen peroxide (100 µmol/L H2O2) was used as a positive control for senescence and to mimic ischemic conditions. Cells were stained for senescence-associated β-galactosidase or processed for immunoblotting and quantitative real-time PCR analyses. In additional experiments, cells were stimulated with ET-1 in the presence or absence of ETA receptor antagonist BQ-123 (20 μmol/L) or ETB receptor antagonist BQ-788 (20 μmol/L). ET-1 stimulation increased β-galactosidase accumulation which was prevented by BQ-123. ET-1 also increased traditional senescence marker p16 protein and pericyte-specific senescence markers, TGFB1i1, PP1CA, and IGFBP7. Furthermore, ET-1 stimulated contractile protein α-SMA and microglial marker ostepontin in high glucose suggesting a shift toward an ensheathing or microglia-like phenotype. In conclusion, ET-1 triggers senescence, alters ETA and ETB receptors, and causes phenotypical changes in BMVPCs under diabetes-like conditions. These in vitro findings need to be further studied in vivo to establish the role of ETA receptors in the progression of pericyte senescence and phenotypical changes in VCID. [ABSTRACT FROM AUTHOR]

The endothelin (ET) system has been implicated to contribute to the pathophysiology of cognitive impairment and stroke in experimental diabetes. Our goals were to test the hypotheses that (1) circulating and (or) periinfarct ET-1 levels are elevated after stroke in both sexes and this increase is greater in diabetes, (2) ET receptors are differentially regulated in the diabetic brain, (3) brain microvascular endothelial cells (BMVEC) of female and male origin express the ETA receptor subtype, and (4) diabetes- and stroke-mimicking conditions increase ET-1 levels in BMVECs of both sexes. Control and diabetic rats were randomized to sham or stroke surgery. BMVECs of male (hBEC5i) and female (hCMEC/D3) origin, cultured under normal and diabetes-mimicking conditions, were exposed to normoxia or hypoxia. Circulating ET-1 levels were higher in diabetic animals and this was more pronounced in the male cohort. Stroke did not further increase plasma ET-1. Tissue ET-1 levels were increased after stroke only in males, whereas periinfarct ET-1 increased in both control and diabetic females. Male BMVECs secreted more ET-1 than female cells and hypoxia increased ET-1 levels in both cell types. There was sexually dimorphic regulation of ET receptors in both tissue and cell culture samples. There are sex differences in the stroke- and diabetes-mediated changes in the brain ET system at the endothelial and tissue levels. Key words: diabetes, stroke, ET-1, sex, brain. On a avance que le systeme endotheline (ET) contribue a la physiopathologie des deficits cognitifs et de l'AVC dans le cadre du diabete experimental. Nous avions pour but de verifier les hypotheses selon lesquelles 1) les taux d'ET-1 en circulation ou en peripherie de l'infarctus augmentent apres un AVC chez les patients des deux sexes et cette augmentation est plus importante en cas de diabete, 2) les recepteurs de l'ET sont regules de maniere differentielle dans le cerveau en cas de diabete, 3) les cellules endotheliales des micro-vaisseaux du cerveau (CEMVC) d'origines male et femelle expriment le recepteur du sous-type ETA et 4) des situations mimant le diabete et l'AVC entrainent une augmentation des taux d'ET-1 dans les CEMVC pour les deux sexes. Nous avons reparti aleatoirement des rats temoins et diabetiques entre des groupes d'interventions chirurgicales factices (<>) et entrainant un AVC. Nous avons expose a la normoxie et a l'hypoxie des CEMVC d'origines male (hBEC5i) et femelle (hCMEC/D3) en cultures mimant une situation normale ou le diabete. Les taux d'ET-1 en circulation etaient plus eleves chez les animaux diabetiques, et ce, de facon plus marquee pour les cellules males. L'AVC n'entrainait pas d'augmentation supplementaire des taux plasmatiques d'ET-1. Apres l'AVC, les taux d'ET-1 tissulaire augmentaient uniquement pour les cellules males, tandis que les taux d'ET-1 en peripherie de l'infarctus augmentaient pour les cellules temoins comme femelles diabetiques. Les CEMVC males secretaient plus d'ET-1 que les cellules femelles et l'hypoxie entrainait une augmentation des taux d'ET-1 dans les deux types cellulaires. On observait une regulation dimorphe des recepteurs de l'ET sur le plan sexuel, et ce, dans les echantillons de tissu comme pour les cellules en culture. Il existe des differences entre les sexes quant aux variations mediees par l'AVC et le diabete dans le systeme ET cerebral aux niveaux endothelial et tissulaire. [Traduit par la Redaction] Mots-cles: diabete, AVC, ET-1, sexe, cerveau., Introduction Diabetes is a major risk factor for neurological diseases including acute ischemic stroke and post-stroke cognitive impairment (Flores-Gomez et al. 2019; Li et al. 2018). Individuals with diabetes also [...]

Diabetes increases the risk and severity of cognitive impairment, especially after ischemic stroke. It is also known that the activation of the endothelin (ET) system is associated with cognitive impairment and microglia around the periinfarct area produce ET-1. However, little is known about the effect of ET-1 on microglial polarization, especially under diabetic conditions. We hypothesized that (i) ET-1 activates microglia to the proinflammatory M-1-like phenotype and (ii) hypoxia/ lipopolysaccharide (LPS) activates the microglial ET system and promotes microglial activation towards the M-1 phenotype in diabetic conditions. Microglial cells (C8B4) cultured under normal-glucose (25 mmol/L) conditions and diabetes-mimicking high-glucose (50 mmol/L) conditions for 48 h were stimulated with ET-1, cobalt chloride (200 [micro]mol/L), or LPS (100 ng/mL) for 24 h. PPET-1, ET receptor subtypes, and M1/M2 marker gene mRNA expression were measured by RT-PCR. Secreted ET-1 was measured by ELISA. A high dose of ET-1 (1 [micro]mol/L) increases the mRNA levels of ET receptors and activates the microglia towards the M1 phenotype. Hypoxia or LPS activates the ET system in microglial cells and shifts the microglia towards the M1 phenotype in diabetic conditions. These in vitro observations warrant further investigation into the role of ET-1-mediated activation of proinflammatory microglia in post-stroke cognitive impairment in diabetes. Key words: diabetes, stroke, ET-1, microglial cells, brain. Le diabete entraine un accroissement du risque et de la gravite des deficits cognitifs, particulierement apres un AVC. On sait aussi que l'activation du systeme endotheline (ET) est associe a des deficits cognitifs et que la microglie de la bordure de l'infarctus produit de l'ET-1. Cependant, on sait peu de choses de l'effet de l'ET-1 sur la polarisation de la microglie, particulierement en cas de diabete. Nous avons pose des hypotheses selon lesquelles i) l'ET-1 entraine une activation de la microglie vers un phenotype pro-inflammatoire s'apparentant a M-1 et ii) l'hypoxie et les lipopolysaccharides entrainent une activation du systeme ET de la microglie et favorise l'activation de la microglie vers un phenotype M-1 en cas de diabete. Nous avons cultive des cellules microgliales (C8B4) en presence de taux de glucose normaux (a 25 mmol/L) et mimant l'hyperglycemie du diabete (a 50 mmol/L) pendant 48 h avec de l'ET-1, du chlorure de cobalt (a 200 [micro]mol/L) ou des lipopolysaccharides (a 100 ng/mL) pendant 24 h. A l'aide de la technique de PCR en temps reel, nous avons mesure l'expression de sous-types de recepteurs de la prepro-ET et de l'ET, ainsi que celle de l'ARNm des genes des marqueurs de M1 et de M2. Nous avons mesure la secretion d'ET-1 a l'aide de la technique d'ELISA. L'ET-1 a dose elevee (a 1 [micro]mol/L) entraine une augmentation des taux d'ARNm des recepteurs de l'ET avec une activation de la microglie vers le phenotype M1. L'hypoxie et les LPS entrainent une activation du systeme de l'ET dans les cellules microgliales et fait tendre la microglie vers le phenotype M1 en situation de diabete. Ces observations in vitro viennent justifier de plus amples recherches quant au role de l'activation de la microglie pro-inflammatoire mediee par l'ET-1 dans le deficit cognitif post-AVC en cas de diabete. [Traduit par la Redaction] Mots-cles: diabete, AVC, ET-1, cellules microgliales, cerveau., Introduction Diabetes induces a state of chronic inflammation and increases the risk of cerebrovascular diseases. Activation of the endothelin (ET) system has been also implicated in ischemic/hypoxic injuries in the [...]

Diabetes increases the risk of poststroke cognitive impairment (PSCI). Greater hemorrhagic transformation (HT) after stroke is associated with vasoregression and cognitive decline in male diabetic rats. Iron chelator deferoxamine (DFX) prevents vasoregression and improves outcomes. Although diabetic female rats develop greater HT, its impact on poststroke cerebrovascularization and cognitive outcomes remained unknown. We hypothesized that diabetes mediates pathological neovascularization, and DFX attenuates poststroke cerebrovascular remodeling and improves neurological outcomes in female diabetic rats. Female control and diabetic animals were treated with DFX or vehicle for 7 days after stroke. Vascular indices, microglial activation, and blood-brain barrier (BBB) integrity were evaluated on day 14. Results from diabetic female rats were partially compared with our previously published findings in male counterparts. Hemin-induced programmed cell death was studied in male and female brain microvascular endothelial cell lines (BMVEC). There was no vasoregression after stroke in either control or diabetic female animals. DFX prevented diabetes-mediated gliovascular remodeling and compromised BBB integrity while improving memory function in diabetes. Comparisons of female and male rats indicated sex differences in cognitive and vascular outcomes. Hemin mediated ferroptosis in both male and female BMVECs. DFX improved survival but had differential effects on ferroptosis signaling in female and male cells. These results suggest that stroke and associated HT do not affect cerebrovascularization in diabetic female rats, but iron chelation may provide a novel therapeutic strategy in the prevention of poststroke memory impairment in females with diabetes via the preservation of gliovascular integrity and improvement of endothelial cell survival. [ABSTRACT FROM AUTHOR]