Endothelin A receptors contribute to senescence of brain microvascular endothelial cells

Abstract

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. Serum-starved human BMVECs (HBEC5i) were incubated with ET-1 (1 µmol/L) in the presence/absence of ETA receptor antagonist BQ-123 (20 µmol/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 β-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 (VE-cadherin) 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.

Keywords: brain; cellules endothéliales; cerveau; endothelial cells; endothelin-1; endothéline 1; senescence; sénescence.

Fig. 1

Schematic representation of cell culture experiments.

Fig. 2

Treatment of BMVECs with an oxidative stress inducer, an inflammatory cytokine, and age-related factor increases senescence associated (SA)- β gal activity. Incubation of BMVECs with a low dose of H₂O₂ (100μM; 3 days), TNF-α (20ng/ml media for 3 days), and Aβ−40 (5μM; for 5 days) increased the SA-β gal staining. Green immunofluorescence indicates the increase in SA-β gal activity. Images were captured at a 20x magnification scale bar is 100 μm (n=3). Data were analyzed with one-way ANOVA, *p,0.05.

Fig. 3

ET-1 increases the ETA receptor protein in BMVECs. ETA and ETB receptor expression levels were measured by RT-qPCR (A&B) and Western blotting (C&D). (A&C) Incubation of BMVECs with ET-1 (1μM) for 16 hours increased protein but not the mRNA level of the ETA receptor and pre-incubation with ETA antagonist BQ-123 prevented this increase. ET-1 treatment did not affect mRNA (B) or protein levels of the ETB receptor. RT-qPCR data are presented fold change while Western blot data are normalized with β-actin and presented as mean ± SEM of percent of control (n=3; in triplicates). Data were analyzed with one-way ANOVA, *p<0.05.

Fig. 4

ET-1 treatment increased the SA-β gal staining which was prevented by the presence of ETA receptor antagonist BQ-123. Cells were treated with ET-1 (1μM) for 16 hours and ETA receptor antagonist (BQ123; 20 μM; treated 30 minutes before ET-1 treatment). Green immunofluorescence indicates the SA-β gal activity. Images were captured at 20x magnification with 2x zoom, scale bar is 50μm (n=3). Data were analyzed with one-way ANOVA, *p<0.05.

Fig. 5

ET-1 treatment increased senescence marker proteins. ETA receptor antagonism reversed this phenomenon. mRNA levels of LIF1 (A), LIF receptor (B), and p21 (C) levels were not affected by ET-1 treatment or ETA receptor inhibition. Senescence marker proteins LIF1 (D), p21 (E), and cyclin D1 (G) but not p16 (F) were increased with ET-1 treatment which was prevented by the ETA receptor blockade. (H) Lamin A/C was significantly decreased with ET-1 treatment and ETA inhibition did not change it. Cells were treated with ET-1 (1μM) for 16 hours and ETA receptor antagonist (BQ123; 20 μM; treated 30 minutes before ET-1 treatment). RT-qPCR data are presented fold change values while Western blot data are normalized with β-actin and presented as percent of control (n=3; in duplicate or triplicates). Data are plotted as mean ± SEM (n=3; in triplicates). Data was analyzed with one way ANOVA, *p<0.05, **p<0.01, ****p<0.0001.

Fig. 5

ET-1 treatment increased senescence marker proteins. ETA receptor antagonism reversed this phenomenon. mRNA levels of LIF1 (A), LIF receptor (B), and p21 (C) levels were not affected by ET-1 treatment or ETA receptor inhibition. Senescence marker proteins LIF1 (D), p21 (E), and cyclin D1 (G) but not p16 (F) were increased with ET-1 treatment which was prevented by the ETA receptor blockade. (H) Lamin A/C was significantly decreased with ET-1 treatment and ETA inhibition did not change it. Cells were treated with ET-1 (1μM) for 16 hours and ETA receptor antagonist (BQ123; 20 μM; treated 30 minutes before ET-1 treatment). RT-qPCR data are presented fold change values while Western blot data are normalized with β-actin and presented as percent of control (n=3; in duplicate or triplicates). Data are plotted as mean ± SEM (n=3; in triplicates). Data was analyzed with one way ANOVA, *p<0.05, **p<0.01, ****p<0.0001.

Fig. 5

ET-1 treatment increased senescence marker proteins. ETA receptor antagonism reversed this phenomenon. mRNA levels of LIF1 (A), LIF receptor (B), and p21 (C) levels were not affected by ET-1 treatment or ETA receptor inhibition. Senescence marker proteins LIF1 (D), p21 (E), and cyclin D1 (G) but not p16 (F) were increased with ET-1 treatment which was prevented by the ETA receptor blockade. (H) Lamin A/C was significantly decreased with ET-1 treatment and ETA inhibition did not change it. Cells were treated with ET-1 (1μM) for 16 hours and ETA receptor antagonist (BQ123; 20 μM; treated 30 minutes before ET-1 treatment). RT-qPCR data are presented fold change values while Western blot data are normalized with β-actin and presented as percent of control (n=3; in duplicate or triplicates). Data are plotted as mean ± SEM (n=3; in triplicates). Data was analyzed with one way ANOVA, *p<0.05, **p<0.01, ****p<0.0001.

Fig. 6

ET-1 decreased the expression of tight junction proteins in BMVECs. (A) mRNA expression of zo-1, VE-cadherin, and claudin-5 were differently regulated in response to ET-1 and ETA antagonism. (B) Protein expression of occludin-1, VE-cadherin, and claudin-5 was decreased with ET-1, while ETA antagonism did not improve it. Cells were treated with ET-1 (1μM) for 16 hours and ETA receptor antagonist (BQ123; 20 μM; treated 30 minutes before ET-1 treatment). RT-qPCR data are presented as fold change values, while Western blot data are normalized with β-actin and presented as percent of control (n=3; in duplicate or triplicates). Data are plotted as mean ± SEM (n=3; in triplicates). Data were analyzed with one-way ANOVA, *p<0.05.