High Glucose and Hypoxia-Mediated Damage to Human Brain Microvessel Endothelial Cells Induces an Altered, Pro-Inflammatory Phenotype in BV-2 Microglia In Vitro

Abstract

Diabetes is strongly linked to the development of Alzheimer's disease (AD), though the mechanisms for this enhanced risk are unclear. Because vascular inflammation is a consistent feature of both diabetes and AD, the cerebral microcirculation could be a key target for the effects of diabetes in the brain. The goal of this study is to explore whether brain endothelial cells, injured by diabetes-related insults, glucose and hypoxia, can affect inflammatory and activation processes in microglia in vitro. Human brain microvascular endothelial cells (HBMVECs) were either treated with 5 mM glucose (control), 30 mM glucose (high glucose), exposed to hypoxia, or exposed to hypoxia plus high glucose. HBMVEC-conditioned medium was then used to treat BV-2 microglia. Alterations in microglia phenotype were assessed through measurement of nitric oxide (NO), cytokine production, microglial activation state markers, and microglial phagocytosis. HBMVECs were injured by exposure to glucose and/or hypoxia, as assessed by release of LDH, interleukin (IL)-1β, and reactive oxygen species (ROS). HBMVECs injured by glucose and hypoxia induced increases in microglial production of NO, tumor necrosis factor-α (TNFα) and matrix metalloproteinase (MMP)-9. Injured HBMVECs significantly increased microglial expression of CD11c and CLEC7A, and decreased expression of the homeostatic marker P2RY12. Finally, bead uptake by BV-2 cells, an index of phagocytic ability, was elevated by conditioned media from injured HBMVECs. The demonstration that injury to brain endothelial cells by diabetic-associated insults, glucose and hypoxia, promotes microglial inflammation supports the idea that the cerebral microcirculation is a critical locus for the deleterious effects of diabetes in the AD brain.

Keywords: Alzheimer’s disease; Endothelial; Inflammation; Microglia; Vascular.

Fig. 1

HBMVEC injury following treatment with high glucose with and without hypoxia HBMVECs were exposed to high glucose (6 h), hypoxia (1 h), or a combination of both (6 h). Cells and supernatant were collected to assess measures of cellular injury: a lactate dehydrogenase (LDH) release as a measure of cell toxicity, b reactive oxygen species (ROS) production measured with DCF-DA, represented as relative fluorescence units (RFU), and (C) IL-1β secretion in the supernatant measured by ELISA. ***p < 0.001 vs. Control, ^###p < 0.001 vs. Glucose, ^p < 0.05 vs. Hypoxia

Fig. 2

HBMVEC injury induces increased nitric oxide production by BV-2 microglia Conditioned media from injured HBMVECs was used to treat BV-2 microglia for 24 h. Supernatant and cell lysate were collected following treatments and used to measure (a) Nitric Oxide (NO) in the supernatant using Griess reagent (n = 12 per group) and (b) expression of iNOS in the lysate by western blot. **p < 0.01 vs. Control, ***p < 0.001 vs. Control, ^###p < 0.001 vs. Glucose, ^p < 0.05 vs. Hypoxia, ^^^p < 0.001 vs. Hypoxia.

Fig. 3

HBMVEC injury induces increased production of inflammatory mediators by BV-2 microglia Conditioned media from injured HBMVECs was used to treat BV-2 microglia for 24 h. Supernatant from BV-2 was collected following treatments. ELISA was used to measure secretion of (a) TNFα and (b) IL-6. (c) The enzymatic activity of MMP-9 was measured by gel zymography (n = 3 per group). ***p < 0.001 vs. Control, ^###p < 0.001 vs. Glucose, ^^^p < 0.001 vs. Hypoxia.

Fig. 4

BV-2 treated with conditioned media from injured HBMVEC show altered expression of microglia activation markers Conditioned media from injured HBMVECs was used to treat BV-2 microglia for 24 h. Western blot was used to assess the expression of microglia markers, (a) CD11c, (b) CLEC7A, and (c) P2RY12. *p < 0.05 vs. Control, **p < 0.01 vs. Control, ***p < 0.001 vs. Control, ^p < 0.05 vs. Hypoxia, ^^p < 0.01 vs. Hypoxia

Fig. 5

BV-2 treated with conditioned media from injured HBMVEC show altered phagocytic ability Conditioned media from injured HBMVEC was used to treat BV-2 microglia for 24 h. BV-2 were then incubated with fluorescently labeled beads for 1 h. Cells were then imaged (EC-C, EC-G, EC-H, EC-G+H left to right) at 10X (a) and 20X (b) and bead uptake per cell was quantified (c) as a measure of phagocytic activity. *p < 0.05 vs. Control, ^###p < 0.001 vs. Glucose.