Lipid Reshaping and Lipophagy Are Induced in a Modeled Ischemia-Reperfusion Injury of Blood Brain Barrier

Abstract

Ischemic-reperfusion (I/R) injury induced a remodeling of protein and lipid homeostasis, under oxidative stress and inflammatory status. Starvation occurring during I/R is a condition leading to autophagy activation, which allows abnormal material clearance or amino acid, or both, and fatty acid (FA) recycling essential for survival. This study investigated the lipid reshaping, peroxidation, and related-signaling pathways, in rat brain endothelial cells (RBE4) subjected to 3 h of oxygen and glucose deprivation (OGD) and restoration of standard condition (I/R in vitro model). Lipids and proteins were analyzed after 1 or 24 h of oxygen and nutrient restoration. Together with the oxidative stress and inflammatory status, I/R injury induced a reshaping of neutral lipids and biogenesis of lipid droplets (LD) with excessive lipid storage. The increase of LC3-II/LC3-I ratio, an autophagy marker, and LC3 co-localization with LD suggest the activation of lipophagy machinery to counteract the cell engulfment. Lipophagy leads to cholesterol ester (CE) hydrolysis, increasing free cholesterol (FC) secretion, which occurred by specific transporters or unconventional exocytosis pathways, or both. Here, we propose that an unconventional spreading of FC and other lipid metabolites may influence the neurovascular unit (NVU) cells, contributing to Blood brain barrier (BBB) alteration or adaptation, or both, to the cumulative effects of several transient ischemia.

Keywords: cholesterol; ischemia; lipid droplets; lipophagy; oxygen and glucose deprivation; vessel disease.

Figure 1

Total polyunsaturated fatty acid (PUFA) content of rat brain endothelial cells (RBE4) cells and of purified phosphatidylcholine (PC) after oxygen and glucose deprivation and restoration (OGD/OgR) treatment for 1 and 24 h. Cells, after OGD/ogR treatment, were collected and lipids were extracted by different mixture of methanol/chloroform. Cell FAs (fatty acids) were measured as FA methyl esters (FAME) by a GC (Agilent Technologies 6850 series II, Santa Clara, CA, US) equipped with flame ionization detector. FAME were quantified using the chromatographic peak area according to the internal standard (IS) method. Histograms represent the most relevant results of whole cell (A), and PC (B) FA composition. The data are normalized to cellular protein content and expressed as mean ± S.E. from three independent experiments, * p < 0.05, ** p < 0.01 vs. control.

Figure 2

Analysis of proteins involved in arachidonic acid (AA) metabolism in ogR. Cells subjected to OGD treatment were harvested in hypotonic solution at 1 h and 24 h ogR. Total homogenate aliquot was collected and membrane-enriched fractions (MEFs) were obtained by ultracentrifugation. Equal amounts of MEF or homogenate samples (as protein) were subjected to SDS-PAGE and WB analysis. Ponceau staining of total lanes was employed to perform band quantification of MEF samples, while total lysates were normalized by β-actin content. Panel (A) represents the % of cPLA2 increment and panel (B) the % of COX-2 protein levels with respect to CTRL. The data are expressed as mean ± S.E. from three independent experiments, * p < 0.05, ** p < 0.01 vs. control.

Figure 3

Evaluation of lipid peroxidation in RBE4 after OGD/ogR. Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) content in control and OGD/ogR RBE4 cells as marker of lipid peroxidation by HPLC- Evaporative light scattering detector (ELSD) system. The data are normalized to cellular protein content (nmol/mg protein) and expressed as mean ± S.E from three independent experiments, ** p < 0.01 vs. control, § p < 0.05 vs. ogR1h.

Figure 4

Triglyceride (TG) content in RBE4 after OGD/ogR. Cells, after OGD/ogR treatment, were collected and lipids were extracted by different methanol/chloroform mixtures. TGs were quantified by HPLC-ELSD system. Histogram represents the mean ± S.E. of TG concentration normalized to protein content in the same sample. * p < 0.05, *** p < 0.001 vs. control, n = 3.

Figure 5

Lipid droplet (LD) staining in RBE4 after OGD/ogR. RBE4 cells were fixed in 4% paraformaldehyde and permeabilized at 4 °C with 0.5% Triton X-100 Hepes Buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Images show LDs stained with BODIPY 493/503, actin filaments stained with phalloidin and nuclei stained with DAPI (blue fluorescence) in control (A), ogR1h (B), and ogR24h (C) RBE4 cells. Fluorescence images were collected under a Nikon Eclypse microscope with immersion objective at 60× magnification.

Figure 6

Autophagy marker analysis in RBE4 after OGD/ogR. Cells subjected to OGD/ogR treatment were harvested in lysis buffer, then equal amounts of homogenate samples (as protein) were analyzed by WB. LC3II/I, p62 and Beclin-1 were detected with specific antibodies and revealed by enhanced chemiluminescence (ECL). Sample were normalized on β-actin immunoreactivity. Histograms represent the % of protein levels respect to control as mean ± S.E. from three independent experiments, * p < 0.05, *** p < 0.001 vs. control; § p < 0.05 vs. ogR1h.

Figure 7

LD increase under OGD/ogR. RBE4 cells were fixed in 4% paraformaldehyde and permeabilized at 4 °C with 0.5% Triton X-100 Hepes Buffer. Then cells were tripled marked with LC3 specific antibody/and secondary antibody Alexa Fluor^® 567-labeled (red fluorescence) to stain autophagy vacuoles (AVs), with BODIPY 493/503 (green fluorescence) to stain LD, and with DAPI for nuclei staining in control (A,D,G,J), ogR1h (B,E,H,K) and ogR24h (C,F,I,L) RBE4 cells. Fluorescence images were collected under a Nikon Eclypse microscope with immersion objective at 60× magnification.

Figure 8

Changes in intracellular and extracellular cholesterol content under OGD/ogR. Cells, after OGD/ogR treatment, were collected and lipids were extracted by different mixture of methanol/chloroform. Free cholesterol (FC) and cholesterol ester (CE) were evaluated by HPLC-ELSD. Panel A represents intracellular CE concentration normalized to protein content. Panel (B) depicts with circle charts the molar percentage distribution of intracellular CE and FC. Panel (C) reports the cell medium FC content normalized for protein of cells seeded in each analyzed plate. Data are reported as mean ± S.E. (A,C) from three independent experiments, * p < 0.05, ** p < 0.01 vs. control; § p < 0.05 vs. ogR1h.

Figure 9

Evaluation of MDR1 protein levels inRBE4 after OGD/ogR. Cells subjected to OGD treatment were harvested in lysis buffer, then equal amounts of homogenate samples (as protein) were subjected to SDS-PAGE and WB analysis. MDR1 was detected with the specific antibody and revealed by ECL. Sample were normalized on β-actin immunoreactivity. Histograms represent the % of protein levels respect to control as mean ± S.E from three independent experiments, * p < 0.05 vs. control.