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Published in final edited form as: Can J Physiol Pharmacol. 2020 Mar 2;98(9):596–603. doi: 10.1139/cjpp-2019-0679

Endothelin-1 (ET-1) Promotes a Proinflammatory Microglia Phenotype in Diabetic Conditions

Yasir Abdul 1,2, Sarah Jamil 1,2, Lianying He 1,2, Weiguo Li 1,2, Adviye Ergul 1,2
PMCID: PMC7483816  NIHMSID: NIHMS1608780  PMID: 32119570

Abstract

Diabetes increases the risk and severity of cognitive impairment, especially after ischemic stroke. It is also known that the activation of ET system is associated with cognitive impairment and microglia around 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 a) ET-1 activates microglia to proinflammatory M-1 like phenotype, and b) Hypoxia/LPS activates the microglial ET system and promotes microglial activation towards M-1 phenotype in diabetic conditions. Microglial cells (C8B4) cultured under normal glucose (25mM) and diabetes-mimicking high glucose (50mM) conditions for 48 hours were stimulated with ET-1, cobalt chloride (200μM) or lipopolysaccharide (100ng/ml) for 24 hours. PPET-1, ET receptor subtypes and M1/M2 marker genes mRNA expression measured by RT-PCR. Secreted ET-1 was measured by ELISA. High dose of ET-1 (1μM), increases the mRNA levels of ET receptors and activates the microglia towards M1 phenotype. Hypoxia or LPS activates the ET system in microglial cells and shifts the microglia towards 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: Diabetes, Stroke, ET-1, microglial cells, Brain

INTRODUCTION

Diabetes induces a state of chronic inflammation and increases the risk of cerebrovascular diseases. Activation of the ET system has been also implicated in ischemic/hypoxic injuries in the adult brain (Loo et al. 2002; Yamashita et al. 1993). It is well established that ET-1, the most potent vasoconstrictor identified to date, contributes to diabetes-mediated vascular dysfunction and remodeling in multiple vascular beds including the cerebral vasculature (Abdelsaid et al. 2014a; Abdelsaid et al. 2014b; Ergul et al. 2014; Matsumoto et al. 2004; Yasir et al. 2016). Experimental data suggests that endothelial ET-1 secretion occurs mainly toward the medial layer and thus the vascular effects observed could be due to mostly paracrine and autocrine functions, rather than endocrine effects (Wagner et al. 1992). Previously, the detrimental role of ET-1 in ischemic injury was mainly attributed to the regulation of cerebrovascular tone via the activation of ETA and ETB receptor subtypes on vascular smooth muscle cells and endothelial cells. However, with the advancement in the field, It has been shown that ET like reactivity increases several fold in cerebrospinal fluid (CSF) and glia after ischemic injuries and increases the expression of both ET receptors on non-vascular cells like neuron and glial cells (Barone et al. 1995; Loo et al. 2002). The authors have reported dramatic increase in the ETB receptor expression in microglia in the damaged hippocampus area (Yamashita et al. 1993; Yamashita et al. 1994) but ET-1 effects on microglia in diabetes remained unclear.

Microglial cells are resident immune cells of the brain. They constantly monitor and influence the neurovascular environment in response to injury and later in repair processes (Mallard et al. 2019; McGeer et al. 1993). Microglia exerts its effects by its phenotypic polarization, from quiescent to M1 or M2 phenotype. Activation of microglia to M1 phenotype is attributed to be damaging by the release of inflammatory cytokines and amplification of inflammation processes and secondary neuronal death, whereas, M2 phenotype of microglia promotes CNS recovery by the release of neurotrophic factors mediating synaptic plasticity and angiogenesis (Brown and Neher 2014; Hu et al. 2015; Olah et al. 2012). Our group has recently reported that there was accumulation of activated microglial cells in the hippocampus of diabetic rats after ischemic stroke (Ward et al. 2018). In another recent study, we have observed that there is an increase in the M1 phenotype population in hippocampus and prefrontal cortex regions of ischemic hemisphere after stroke in diabetes (Jackson et al. 2019). Based on these observations, current study was designed to test the hypotheses that 1) ET-1 can activate the microglial cells to M1 phenotype, and 2) Hypoxia/inflammation can trigger the M1 phenotype in microglial cells in diabetic conditions.

MATERIALS AND METHODS

Microglial Cell Culture and Treatments:

Experiments were performed using immortalized mouse microglial cells (C8B4; ATCC, CRL2540), cultured in Dulbecco’s Modified Eagles Media (DMEM; Corning, Manassas, VA, USA) under normal glucose (25mM) and diabetes mimicking high glucose (50mM) conditions for 48 hours. Cells were stimulated with 10nM, 100nM and 1μM ET-1 (Sigma Aldrich) or 200μM cobalt(II)chloride hexahydrate (Sigma Aldrich) to mimic the hypoxia or lipopolysaccharide (LPS, 100ng/ml; Sigma Aldrich) as positive control for 24 hours.

Quantitative RealTime PCR (qRT-PCR):

Microglial cells were lysed in RNA lysis buffer and RNA was isolated using SV Total RNA isolation system (Promega, USA). Quality and quantity of extracted RNA was assayed using a Nanodrop instrument (NanoDrop Technologies, Wilmington, DE). iScript cDNA synthesis kit (cat #1708891, BioRad, Foster City, CA) was used to reverse transcribe equal quantities of total RNA following the manufacturer’s instructions. Primers were custom designed from Invitrogen (Thermo Fisher Scientific). The sequences of primers used in the study are listed in Table 1. qRT-PCR was performed using iScript Reverse Transcription super mix (cat #1708840, Biorad, Foster City, CA) and StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) as per the manufacturer’s protocol. The relative gene expression was analyzed by the delta-delta Ct method using GAPDH as endogenous control gene and normalized to the respective control group.

Table.1.

List of primer sequences used for RTqPCR analysis of genes.

Gene Forward Reverse NCBI Reference
ppET-1 5’-GTCTTGGGAGCCGAACTCAG-3’ 5’-AACCTCCCAGTCCATACGGT-3’ NM_010104.4
ETA 5’-CCCTCTTCACTTAAGCCGCA-3’ 5’-TGCCAGGTTAATGCCGATGT-3’ NM_010332.2
ETB 5’-ACCTGCGAAATGCTCAGGAA-3’ 5’-TGTCTTGGCCACTTCTCGTC-3’ NM_001276296.1
TNF-α 5’-CCACCACGCTCTTCTGTCTA-3’ 5’-AACTGATGAGAGGGAGGCCA-3’ NM_013693.3
IL-17 5’-GACGCGCAAACATGAGTCC-3’ 5’-TTTGAGGGATGATCGCTGCT-3’ NM_010552.3
IL-10 5’-TCCCTGGGTGAGAAGCTGAA-3’ 5’-GCTCCACTGCCTTGCTCTTAT-3’ NM_010548.2
CD-206 5’-GGAGGACTGCGTGGTTATGAA-3’ 5’-TTGTCTGCACCCTCCGGTA-3’ NM_008625.2
RPS-13 5’-GTGCGGCTTGATTTCCTGTG-3’ 5’-CACGTCGTCAGACGTCAACT-3’ NM_026533.3
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ppET-1, Preproendothelin 1; ETA, Endothelin receptor type A; ETB, Endothelin receptor type B; TNF-α, Tumor necrosis factor-alpha; IL-17, Interleukin 17A; IL-10, Interleukin 10; CD206, Mannose receptor, C type 1; RPS-13, Ribosomal protein S-13.

Western Blot Analysis:

Expression of ETA and ETB receptors was measured by immunoblotting. Briefly, equivalent amounts of cell lysates (20 μg protein/lane) were loaded onto 10% SDS-PAGE, proteins separated, and proteins transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin followed by incubation for 12 hours at 4ºC with primary antibody anti-ETA receptor (ab85163, Abcam) or anti-ETB receptor (AER002, Alomone labs) at 1:1000 dilution or anti β-actin at 1:000 dilution. After washing, membranes were incubated for 1 hour at 20ºC with appropriate secondary antibodies (horseradish peroxidase [HRP]-conjugated; dilution 1:5000). Pre-stained molecular weight markers were run in parallel to identify the molecular weight of proteins of interest. For chemiluminescent detection, the membranes were treated with an enhanced chemiluminescent reagent and the signals were monitored on Amersham imager 680 (GE Healthcare Bio-Sciences Corp., Marlborough, MA). Relative band intensity was determined by densitometry on Image-J and normalized with β-actin protein.

Immunofluorescence:

Microglial cells grown on slides were treated with ET-1 (100nM or 1μM) for 24 hours followed by fixation with 4% paraformaldehyde for 15 min, and subsequently washed with TBS followed by treatment with 0.2% Triton X-100 for 3 min. After washing, cells were blocked by 5% BSA for 1 h at room temperature. Cells were then incubated with anti-ETA (AER-001, Alomone Labs, Israel), and ETB (AER-002, Alomone Labs, Israel) antibody at a 1:100 dilution in 0.2% BSA for 3 h at room temperature. Cells were washed and incubated with AlexaFlour 488 conjugated secondary antibody (anti-rabbit; Jackson Immuno Research Laboratories, Inc., West Grove, PA) at a 1:500 dilution at room temperature for 1 h. Negative control slides were incubated with 0.2% BSA in place of the primary antibody (not shown). Slides were imaged on Olympus IX73 microscope (Olympus Corporation, Japan).

ET-1 ELISA:

Media was collected at the termination of experiment and concentrated to a volume of 500 μl and ET-1 was measured using commercially available ELISA kit from R&D Systems (QuantiGlo ELISA) following the manufacturer’s instruction. Results are normalized with total protein in cell lysate and expressed as pg/mg protein.

Data Analysis:

One-way ANOVA was used to analyze data with multiple groups and followed by a Tukey’s post-hoc comparison. Data was expressed as Mean ± SEM. p<0.05 was considered significant.

RESULTS

ET-1 treatment increases ET receptors on microglial cells

ET-1 increased the gene expression of both ETA and ETB receptors. While ET-1 upregulated the ETA receptor mRNA in a dose-dependent manner (Fig. 1A), at the protein level, there was no significant change in the ETA expression by immunoblotting (Fig. 1B) but immunohistochemistry with the same antibody showed a marked increase with 100nM and 1μM ET-1 (Fig. 1C). All three doses of ET-1 appeared to increase the ETB receptor gene expression to a similar level, however, significance was not achieved due to high variability (Fig. 2A). On the other hand, the ETB receptor protein was significantly increased at 100nM and 1μM doses as detected by immunoblotting (Fig. 2B) and immunohistochemistry (Fig. 2C). Interestingly, higher dose of ET-1 showed pronounced nuclear translocation of these receptors.

Fig. 1.

Fig. 1

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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.

Fig. 2

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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.

ET-1 treatment triggers M1 phenotype in microglial cells

Microglial cells are known to switch its phenotype like macrophages. Microglial cells switch to either M1, a proinflammatory phenotype or M2 an anti-inflammatory phenotype from a surveillance state. ET-1 increased the mRNA expression of IL-17, an M1 marker gene but its effect on another M1 marker, TNF-α, was not as robust (Fig. 3A and B). ET-1 treatment significantly decreased M2 marker gene CD206 and there was a trend (p=0.1) for lower IL-10 after ET-1 stimulation (Fig. 3 C and D).

Fig. 3.

Fig. 3

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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)

Diabetic conditions and hypoxia activate the ET system in microglial cells

Diabetes is known to create a chronic inflammatory condition while hypoxia is a major contributor to neurodegenerative diseases. Thus, we next assessed the effect of these conditions on the ET system in microglial cells. LPS was also used as a trigger for the inflammation. Hypoxia did not increase ET-1 secretion either in normal or diabetic conditions. However, LPS significantly increased ET-1 secretion in both normal and diabetic conditions (Fig. 4A). One-way ANOVA analysis showed a significant difference between preproET-1 mRNA levels between the groups under normal glucose conditions and this was due to increased PPET-1 expression with LPS treatment (post-hoc analysis p<0.05 vs NG or NG-hypoxia). In diabetes mimicking conditions, both hypoxia and LPS (post-hoc p<0.01 vs HG) increased the PPET-1 mRNA (Fig. 4B). There was no change in ETA and ETB expression levels under normal growth conditions. While it did not reach to significance for ETA receptors (p=0.07), ETB receptors were upregulated with hypoxia as well as LPS in diabetic conditions (Fig. 4C and D).

Fig. 4.

Fig. 4

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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).

Diabetic conditions and hypoxia trigger M1 phenotype in microglial cells

Since we observed the activation of the ET system under high glucose and hypoxia conditions in microglial cells, we decided to look at the phenotype of these cells in terms of M1/M2 ratio. The mRNA expression of TNF-α was upregulated under hypoxia in normal conditions but surprisingly it did not increase in diabetic conditions. LPS treatment significantly increased TNF-α mRNA expression in both normal and diabetic conditions (Fig. 5A). Expression of IL-17 mRNA was also different between the three groups and this effect was more pronounced under high glucose conditions (Fig. 5B, p<0.05 vs NG and HG respectively). Expression of IL-10 mRNA was unchanged under normal conditions. However, in diabetes-like conditions, there was a robust difference between the groups stemming from an unexpected hypoxia-mediated increase in IL-10 (Fig. 5C). Expression of CD206 mRNA was significantly reduced with hypoxia and LPS both under normal and diabetic conditions (Fig. 5D).

Fig. 5.

Fig. 5

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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).

DISCUSSION

The current study in a cell culture model provides new evidence that 1) ET-1 upregulates the mRNA as well as protein expression of both ETA and ETB receptors in microglia, 2) ET-1 increases the expression of M1 phenotype marker genes while decreasing the M2 phenotype marker genes, 3) Hypoxia increases the expression of both ETA and ETB receptor genes in diabetic conditions, 4) Hypoxia also promotes M1 phenotype while downregulating the M2 phenotype in both normal and diabetic conditions, 5) LPS treatment increases mRNA expression of propre-ET-1 gene as well as ET-1 secretion in media of both control and diabetic conditions, while the expression of ETA and ETB genes are increased only in diabetic conditions, and 6) LPS promotes M1 phenotype while downregulating the M2 phenotype in both normal and diabetic conditions. These findings suggest the involvement of the ET system in complex regulation of microglial polarization in inflammatory disease states that are associated with neurological complications.

Diabetes is one of the most common metabolic disorders that increases the risk and severity of ischemic stroke and poor recovery (Ergul et al. 2012). Exacerbated and sustained neuroinflammation is believed to contribute to this outcome and microglial cells have gained significant attention for their regulatory role in the neuroinflammatory response as the resident immune cells of the brain (Eldahshan et al. 2019). Microglia are watchman of the brain, constantly monitoring the microenvironment and responding to injury. There are multiple microglial phenotypes that constantly switch from one form to another in a very dynamic manner (Eldahshan et al. 2019). It is believed that M1 like microglia are proinflammatory and involved in the generation of free radicals and cytokines leading to tissue injury and neuronal degeneration. Whereas, M2 like phenotype microglia promote CNS recovery by promoting synaptic plasticity (Olah et al. 2012; Paolicelli et al. 2011). Clinical as well as experimental models of ischemic stroke have revealed that microglial activation status changes during different stages of ischemic stroke (Loo et al. 2002). Our group has recently reported that activated microglia are increased in ischemic hippocampus and contribute to poststroke cognitive impairment in an animal model of ischemic stroke in diabetes (Ward et al. 2019; Ward et al. 2018). Further, it has been reported that stroke in diabetes promote the M1 phenotype in ischemic hemisphere (Jackson et al. 2019). At the same time, the ET system is found to be upregulated after ischemic stroke and it is more pronounced in diabetes (Coucha et al. 2018; Li et al. 2018). Since ET receptors are found in microglial cells and upregulated in the periinfarct area after stroke (Li et al. 2010; Yamashita et al. 1994), in the current study we took an in vitro experimental approach to assess the direct effect of ET-1 on microglial ET receptor expression and polarization. Furthermore, based on our recent data that circulating and/or periinfarct ET-1 levels are upregulated in male but not in females after stroke in diabetes (under consideration for publication as ET-16 Conference Proceedings), we were interested in investigating the ET-1 effects in microglial cells isolated from male animals. Our findings show that ET-1 upregulates mainly the ETB receptor expression and this is associated with an increase in M1 and a decrease in M2 phenotype marker genes, collectively suggesting that the ETB receptor subtype mediates the M1 polarization.

Diabetes creates a constant inflammatory condition in the brain (Mansur et al. 2014) and is known to activate microglia. Additionally, a secondary insult like ischemic stroke can exacerbate the inflammation within the brain (Ward et al. 2018) and promote M1 phenotype microglial activation (Jackson et al. 2019). It has been also reported that following middle cerebral artery occlusion in rats, there is rapid increase in expression of ETA and ETB receptors in neurons and microglia respectively (Hino et al. 1996; Loo et al. 2002). Thus, the next step of experiments investigated the changes in the microglial ET system and polarization markers under hypoxic and diabetes-mimicking high glucose and inflammatory conditions. We observed that ET-1 secretion into media was not increased in response to hypoxia, while mRNA expression of PPET-1 gene was slightly upregulated in diabetic conditions. Expression of ET receptors was upregulated in response to hypoxia and LPS in diabetic conditions. It has been previously demonstrated that LPS and endothelins can synergistically enhance the expression of iNOS (inducible nitric oxide synthase), a marker of M1 phenotype, in glial cell culture and this effect was mediated through the ETB receptors (Oda et al. 1997). Hypoxia in diabetic conditions increased the IL-17 mRNA expression while decreased the CD206 mRNA expression indicating the M1 polarization of microglial cells and activation towards proinflammatory phenotype. Based on our findings that the ET-1 stimulation and hypoxia/high glucose challenge mediate similar effects on the expression of microglial ET receptors and polarization markers, the next step would be to design future experiments to probe the role of each receptor subtype under hypoxia/high glucose by using molecular and pharmacological interventions and fully characterize the microglia polarization with flow-cytometry building upon the gene expression studies conducted in the current study.

In summary, the findings of the current study demonstrate that ET-1 can activate the microglial ET receptors and promote M1 phenotype polarization. Hypoxia in diabetes-mimicking conditions also activate the microglial ET system and promote the M1 phenotype. These findings are important because while a number of studies have shown that ETA/ETB receptor antagonism improves post stroke recovery (Gupta et al. 2005; Matsumoto et al. 2004; Zhang et al. 2008), recent findings demonstrate that ETB agonism also provides neuroprotection after ischemic stroke (Briyal et al. 2019) and the ETB receptors are required for neurogenesis and angiogenesis (Gulati 2016). Thus, identifying the impact of ET-1 and ETA/ETB receptor agonism/antagonism on microglial dynamics in vivo and in vitro can help in further defining the complex role of the ET system on the modulation of stroke recovery especially in comorbid conditions like diabetes.

ACKNOWLEDGEMENT

Funding This study was supported by Veterans Affairs (VA) Merit Review (BX000347), VA Senior Research Career Scientist Award (IK6 BX004471), National Institute of Health (NIH) R01NS083559 and R01 NS104573 (multi-PI, Susan C. Fagan as co-PI) to Adviye Ergul; and Diabetic Complications Research Consortium DiaComp awards 17AU3831/18AU3903 (DK076169/115255) to Dr. Weiguo Li.

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