Responses of endothelial cell and astrocyte matrix-integrin receptors to ischemia mimic those observed in the neurovascular unit

Abstract

Background and purpose: Apposition of endothelial cells and astrocyte foot processes to the basal lamina matrix is postulated to underlie the cerebral microvessel permeability barrier. Focal cerebral ischemia induces rapid loss of select matrix-binding integrins from both cell compartments in the nonhuman primate. This study is the first to examine the conditions underlying integrin loss from these cell-types during ischemia in vitro and their relation to the changes in vivo.

Methods: The impact of normoxia or standardized oxygen-glucose deprivation on integrin expression by murine primary cerebral endothelial cells and astrocytes grown on matrix substrates (collagen IV, laminin, and perlecan) of the basal lamina were quantitatively assessed by flow cytometry.

Results: Endothelial cell expression of the beta1 and alpha 5 subunits significantly increased on all matrix ligands, whereas astrocytes displayed modest significant decreases in alpha 5 and alpha 6 subunits. Oxygen-glucose deprivation produced a further significant increase in subunit beta1 expression by both cell types, but a clear decrease in both alpha1 and alpha 6 subunits by murine astrocytes.

Conclusions: Ischemia in vitro significantly increased endothelial cell beta1 expression, which is consistent with the increase in beta1 transcription by microvessels peripheral to the ischemic core. The loss of alpha1 and alpha 6 integrins from murine astrocytes is identical to that seen in the nonhuman primate in vivo. These findings establish both isolated murine cerebral endothelial cells and astrocytes as potential integrin response cognates of microvascular cells of the neurovascular unit in primates, and allow determination of the mechanisms of their changes to ischemia.

Figure 1

Murine astrocytes (from mixed glial culture; A) and endothelial cells (B through E) are shown in culture by phase contrast microscopy. Confluent endothelial cells express the tight junction proteins claudin-5 (C), occludin (D), and ZO-1 (E), detected by immunofluorescence. Phase bright cells in panel A are microglial cells. Magnification bars: A and B=50 μm; C through E=25 μm.

Figure 2

Influence of matrix protein substrate and OGD on endothelial and astrocyte integrin expression. A through D, Endothelial cells and astrocytes were cultured for 2 days on plastic (black line) or laminin (shaded area) before expression of the β1 and α5 integrin subunits was quantified by flow cytometry. E through H, Endothelial cells and astrocytes grown on laminin were exposed to normoxia (black line) or ischemia (shaded area) for 18 hours before expression of the β1 and α6 integrin subunits was quantified by flow cytometry. Note that laminin increased β1 and α5 integrin expression by endothelial cells but not astrocytes. OGD promoted increased β1 expression on endothelial cells and astrocytes, but reduced α6 expression specifically on astrocytes.

Figure 3

The influence of basal lamina matrix proteins on integrin expression by primary endothelial cells and astrocytes. Both cell populations were cultured on collagen I (black), collagen IV (white), fibronectin (hatched), laminin (gray), or HSPG/perlecan (reverse hatched). Subunit expression by flow cytometry is recorded as the mean percentage change (±SD) relative to uncoated plastic (control) of 3 experiments each. Significant changes (horizontal bars, P<0.05) were observed for β1 and α5 on endothelial cells, and α6 on astrocytes.

Figure 4

The effects of OGD on integrin expression by primary endothelial cells and astrocytes. Primary endothelial cells and astrocytes were cultured on collagen IV (white), laminin (gray), or HSPG/perlecan (reverse hatched). OGD induced significant (horizontal bars, P<0.05) increases in the integrin β1 subunit expression on endothelial cells and astrocytes, and decreases of both integrins α1 and α6 on astrocytes. Data are the mean±SD of 3 experiments.

Figure 5

Transcription levels of integrin β1 by primary astrocytes after OGD compared with paired cultures exposed to normoxia by real time RT-PCR (significant on plastic, P=0.002). Data are the mean±SD of 3 experiments each.