Diabetes-related sex differences in the brain endothelin system following ischemia in vivo and in human brain endothelial cells in vitro

Abstract

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.

Keywords: AVC; ET-1; brain; cerveau; diabetes; diabète; sex; sexe; stroke.

Fig. 1.

Circulating and peri-infarct brain ET-1 levels are higher in diabetic male animals. Effect of stroke and diabetes on male plasma and brain tissue ET-1 levels in male (A and D) and female (B and E) animals are analyzed by two-way ANOVA for each sex. Female and male plasma (C) and brain tissue (F) comparisons were made only in the diabetic cohorts by two-way ANOVA. Significance is indicated on each graph and when there is an interaction or a trend for interaction, full ANOVA tables are shown on the graphs.

Fig. 2.

Peri-infarct PPET-1 and ET receptor expression is differentially regulated by sex and stroke. Fold changes in PPET-1, ETA and ETB mRNA expression are shown in male (A-C) and female (D-F) animals. Given the large variability and a small sample size, statistical comparisons are not marked.

Fig. 3.

Hypoxia increases ET-1 levels to a greater extent in BMVECs of male origin. Diabetes and stroke-mimicking conditions were achieved by culturing cells in high glucose and palmitate (HG+P) containing growth media and exposing to hypoxia induced by CoCl₂ treatment. Changes in ET-1 levels and ETA and ETB mRNA expression are shown in male (A-C) and female (D-F) cells. Two-way ANOVA was performed for each sex (normoxia vs hypoxia) x (NG vs HG+P). Significance is indicated on each graph. Results are shown as mean ± SEM of 4 individual experiments performed in multiple replicates. NG: normal glucose, HG+P: high glucose + palmitate

Fig. 4.

BMVECs of male origin exhibit greater signal for ETA receptors as compared to female cells. Representative images from 3 individual experiments are shown. Images were captured at a magnification of 20x. Data was not quantified. NG: normal glucose, HG+P: high glucose + palmitate

Fig. 5.

ET-1 decreases viability of both cell lines whereas hypoxia attenuates survival only in male BMVECs. Cumulative data for area under the curve (AUC 24 h) for male and female cells is shown in Panels A and D, respectively. ETA receptor blockade with BQ-123 prevents ET-1-mediated decrease in viability in female but not male cells. Representative 28 h tracing of cell viability is shown in Panels B and E and cumulative data is given on Panels C and F. Data was analyzed by one-way ANOVA and results are shown as mean ± SEM of 3 individual experiments performed in triplicate.

Fig. 6.

Hypoxia decreases migration of male but not female BMVECs. Representative images of for male (A) and female (B) cells at the start (0 h) and end (24 h) of a scratch assay are shown on top and cumulative data expressed as % of the distance covered by the migrating cells at the bottom for each cell line. Scratched areas are indicated by lines. Two-way ANOVA was performed for each sex (normoxia vs hypoxia) x (NG vs HG+P). Full ANOVA table indicating interaction is shown on the graphs. Results are shown as mean ± SEM of 3 individual experiments performed in triplicate.