Endothelin-1 (ET-1) promotes a proinflammatory microglia phenotype in diabetic conditions

Abstract

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 μmol/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 μmol/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.

Keywords: AVC; ET-1; brain; cellules microgliales; cerveau; diabetes; diabète; microglial cells; stroke.

Fig. 1

ET-1 increased the gene expression of ETA receptor. ET-1 upregulated the ETA receptor mRNA in a dose-dependent manner (A). At the protein level, there was no significant change in the ETA expression measured by immunoblotting (B) but immunohistochemistry (C) with the same antibody showed a marked increase with 100nM and 1μM ET-1. One-way ANOVA with post hoc multiple comparisons were performed on all the data sets. qRTPCR data is expressed as fold change from controls (n=3–4, in triplicates) and protein expression was normalized with control (n=3, in triplicates). Green, FITC showing receptor expression, DAPI, blue used for nuclear staining. Images were captured at 20x magnification.

Fig. 2

ET-1 increased the ETB receptor protein expression. ET-1 increases ETB receptor gene (A) expression to a similar level, however, not significant due to high variability. On the other hand, the ETB receptor protein was significantly increased at 100nM and 1μM doses as detected by immunoblotting (B) and immunohistochemistry (C). One-way ANOVA with post hoc multiple comparisons were performed on all the data sets. qRTPCR data is expressed as fold change from controls (n=3–4, in triplicates) and protein expression was normalized with control (n=3, in triplicates). Green, FITC showing receptor expression, DAPI, blue used for nuclear staining. Images were captured at 20x magnification. Experiment was repeated in three separate set of samples.

Fig. 3

ET-1 treatment polarizes the microglial cells towards the M1 phenotype. mRNA expression of M1 phenotype markers (TNF-α and IL-17 genes) were upregulated with 1μM ET-1 dose (A and B). However, mRNA expression of the M2 phenotype marker genes (IL-10 and CD206) were downregulated (C and D). One-way ANOVA with post hoc multiple comparisons were performed on all the data sets. qRTPCR data is expressed as fold change from controls (n=3–4, in triplicates)

Fig. 4

Hypoxia in diabetes mimicking conditions activate the microglial ET system. Diabetes and hypoxic conditions were achieved by culturing cells in high glucose (50mM) containing growth media and exposing to CoCl₂ (200μM). Changes in secreted ET-1 levels (A) were measured by ELISA and expression of prepre-ET-1 (B), ETA (C) and ETB (D) were genes measured by RT-PCR. LPS (100ng/ml) was used as positive control. One-way ANOVA with post hoc multiple comparisons were performed on all the data sets. Data is expressed as fold change from controls (n=3–4, in triplicates).

Fig. 5

Hypoxia polarizes the microglial cells towards M1 phenotype in both normal and diabetes mimicking conditions. mRNA expression of M1 phenotype genes (TNF-α in Panel A and IL-17 in Panel B) were upregulated with hypoxia (CoCl₂; 200μM). While, mRNA expression of M2 phenotype markers IL-10 (C) and CD206 (D) genes were downregulated. LPS (100ng/ml) was used as a positive control. It upregulated M1 and downregulated M2 phenotype marker gene mRNA expression in both normal and diabetic conditions. One-way ANOVA with post hoc multiple comparisons were performed on all the data sets. qRTPCR data is expressed as fold change from controls (n=3–4, in triplicates).