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. 2016 Apr;22(4):291-7.
doi: 10.1111/cns.12503. Epub 2016 Feb 4.

Hyperglycemia Increases the Production of Amyloid Beta-Peptide Leading to Decreased Endothelial Tight Junction

A-Ching Chao  1 , Tai-Chi Lee  2 , Suh-Hang Hank Juo  2 , Ding-I Yang  3
Affiliations

Hyperglycemia Increases the Production of Amyloid Beta-Peptide Leading to Decreased Endothelial Tight Junction

A-Ching Chao et al. CNS Neurosci Ther. 2016 Apr.

Abstract

Aims: Amyloid beta-peptide (Aβ), the main component of senile plaques in the Alzheimer's disease (AD) brains, is generated from sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretase. Hyperglycemia in diabetes may compromise barrier integrity in endothelial cells (ECs). However, the roles of endothelial APP in response to high glucose (HG) remain to be delineated. The aims of this study were to test whether HG may increase Aβ secretion, thereby leading to heightened paracellular permeability in ECs.

Methods: We determined the effects of HG on production of Aβ, expression of full-length APP, intercellular permeability, and expression levels of specific junctional proteins in human umbilical vein endothelial cells (HUVECs).

Results: HG at 30 mM significantly stimulated expression of full-length APP accompanied by heightened secretion of Aβ1-42, increased paracellular permeability, and attenuated expression of zona occluden-1 (ZO-1), claudin-5, occludin, and junctional adhesion molecule (JAM)-C in HUVECs; all of which were abolished by the γ-secretase inhibitor BMS299897. Exogenous application of Aβ1-42, but not the reverse peptide Aβ42-1, was sufficient to downregulate the expression of the same junction proteins.

Conclusion: Hyperglycemia enhances APP expression with increased Aβ production, which downregulates junctional proteins causing increased intercellular permeability in ECs.

Keywords: Alzheimer's disease; Amyloid precursor protein; High glucose; Human umbilical vein endothelial cells; Junctional permeability.

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1
Figure 1
Effects of HG on secretion of Aβ1‐42 and expression of full‐length APP. (A) HUVECs were exposed to 5 mM glucose (NG) or 30 mM glucose (HG) with or without 100 nM γ‐secretase inhibitor BMS299897 for 72 h. The Aβ1‐42 in the culture medium was quantitatively determined by ELISA. Mean ± SEM from three independent experiments. *Denotes P < 0.05 versus NG control; # denotes P < 0.05 versus HG without BMS299897. (B) HUVECs were treated with 5 mM glucose (NG), 30 mM glucose (HG), or 30 mM glucose with 100 nM BMS299897 (HG + I) for indicated times (in days) before detection of full‐length APP by Western blotting. Representative blots from five independent experiments are shown. The lower panel shows the quantitative results of intensities of both bands representing various APP isoforms. *Denotes P < 0.05 as compared to HUVECs exposed to NG for 3 day without γ‐secretase inhibitor BMS299897. Both # and § also denote P < 0.05.
Figure 2
Figure 2
HG‐mediated impairments of vascular permeability in HUVECs. (A) HUVECs were cultured for 72 h to reach full confluence before the monolayer cells were exposed to 5 mM glucose (NG), 30 mM glucose (HG), or 30 mM glucose with 100 nM γ‐secretase inhibitor BMS299897 (HG + I) for additional 72 h. Permeability assay was then performed to quantitatively determine the intercellular passage of FITC‐conjugated dextran. The fluorescence intensity was expressed as relative luminescence units (RLU). Mean ± SEM from three independent experiments. *Denotes P < 0.05 versus NG control; # denotes P < 0.05 versus HG without the γ‐secretase inhibitor BMS299897. (B) Morphological alterations of HUVECs cultured under HG condition were observed. The experimental conditions were the same as described above in (A) except bright‐field images were acquired under a microscope. Scale bar = 100 μm. Note the abnormal morphology of HG‐treated cultures showing enlarged gaps, as compared to the normal pebble‐like morphology with minimal gaps in NG‐treated cultures, that was in part reversed by BMS299897. (C) HUVECs were treated with 5 mM glucose (NG) or 10–50 mM glucose (HG) without or with 100 nM BMS299897 for 72 h. DMSO (0.2%) was included to serve as a vehicle control for BMS299897. Cell survival was determined by the WST‐1 assay. Mean ± SEM from three independent experiments. *Denotes P < 0.05 versus NG control. Note that only HG at 50 mM, but not at lower glucose concentrations, caused significant cytotoxicity; in addition, under NG condition BMS299897 alone was without effects in causing cell death.
Figure 3
Figure 3
Effects of HG and exogenous Aβ1‐42 on the expression of junctional proteins at mRNA levels. (A) HUVECs were exposed to 5 mM glucose with 0.2% DMSO (NG + DMSO), 5 mM glucose with 100 nM BMS299897 (NG + I), 30 mM glucose with 0.2% DMSO (HG + DMSO), or 30 mM glucose with 100 nM BMS299897 (HG + I) for 72 h before RNA extraction and real‐time RT‐PCR to determine the expression levels of the indicated junctional proteins. Mean ± SEM from three independent experiments. Both * and # denote P < 0.05 versus “NG + DMSO” group; § denotes P < 0.05 versus “HG + DMSO” group. (B) HUVECs were treated with 20 μM Aβ1‐42, or the biologically inactive reverse peptide Aβ42‐1 that served as a negative control, for 24 h before real‐time RT‐PCR. Mean ± SEM from three independent experiments. *Denotes P < 0.05 versus the control cultures treated with Aβ42‐1.
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