Elsevier

Regulatory Peptides

Volume 123, Issues 1–3, 15 December 2004, Pages 77-83
Regulatory Peptides

Effects of hypoxia on endothelial/pericytic co-culture model of the blood–brain barrier

https://doi.org/10.1016/j.regpep.2004.05.023Get rights and content

Abstract

The blood–brain barrier (BBB) is composed of endothelial cells, pericytes and astrocytic foot processes. Most research for the in vitro BBB is performed endothelial cells with or without astrocytes. Hypoxia damage to the BBB induces vasogenic brain edema. We have generated a new model of the BBB with brain endothelial cells and pericytes and have examined the effects of hypoxia using this model.

Brain microvascular endothelial cells and pericytes were isolated from three-week-old male Wister rats using enzyme and mechanical homogenization. Three models (A: only endothelial monolayer, B: endothelial monolayer with pericytes non-contact condition, and C: contact condition) were made by culturing these cells using Transwell co-culture system and were exposed to hypoxic condition. We evaluated barrier function with transendothelial electrical resistance (TEER) and permeability of Evans blue-albumin and sodium fluorescein.

The tightest barrier was observed in the endothelial/pericytic contact model. Despite hypoxia-induced disruption of the barrier in endothelial monolayer and non-contact co-culture models, a minimum of dysfunction was seen in the contact co-culture model. Therefore, it is considered that pericytes effect on the endothelia by secreting factors or through a gap junction.

In short, pericytes induce endothelial maturation and a tighter barrier function, which supports the function against the hypoxic injury. Intercellular communication might be important to keep the BBB functional and stabilize in hypoxia.

Introduction

The blood–brain barrier (BBB) is formed by epithelial-like high resistance tight junctions within the endothelium of capillaries perfusing the vertebrate brain. Tight junctions are created by several integral membrane proteins, including occludins, claudins and associated cytoplasmic proteins, e.g., zonula occludens (ZO)-1 or ZO-2. While BBB permeability is controlled by the biochemical properties of the plasma membranes of the capillary endothelial cells, the overall brain microvascular biology is a function of the paracrine interactions between the capillary endothelium and the other two major cells comprising the microcirculation of brain, i.e., the capillary pericyte and the astrocyte foot process. These cells compose 99% of the abluminal surface of the capillary basement membrane in brain [1], [2], [3], [4]. Pericytes may serve multiple functional roles in the BBB [5]. Contraction (and reciprocal relaxation) appears to be the mechanism by which pericytes regulate microvascular blood flow, similar to the smooth muscle of larger vessels [6], [7]. The regulation of endothelial cells has been suggested to control vessel growth and contribute to vascular stability [8]. Finally, macrophage activity participates in immune responses in the central nervous system [9].

Continuous microvascular endothelial cells are essential to the integrity of the BBB, and many pathological conditions like local ischemia or brain tumors lead to opening of the BBB. These openings are in through transcellular or paracellular pathways, which may lead to the development of vasogenic brain edema. Until now, astrocytes have been shown to have a protective effect on BBB disruption following ischemia/reperfusion probably by a radical scavenging mechanism [10]. However, the role of pericytes in formation or maintenance of BBB remains to be elucidated, especially in pathological conditions leading to BBB disruption, such as hypoxia/ischemia. The in vitro model of the BBB chosen for these studies consisted of brain microvessel endothelial cells cultured alone, with pericytes grown in the bottom of 12-well plates and pericytes grown opposed to the endothelial cells on the Transwell filter. The functional alterations in BBB permeability under hypoxia were evaluated by measuring transendothelial electrical resistance (TEER) and permeability of sodium fluorescein and Evans blue-albumin.

Our results provide evidence that pericytes grown in contact with endothelial cells, but not in non-contact model, result in both a tighter endothelium (increased electrical resistance and decreased permeability) and increased protection from hypoxic injury.

Section snippets

Endothelial cell isolation

Primary culture of rat brain endothelial cells was prepared from 3-week-old Wistar rats [11]. Meninges was carefully removed from the forebrain and gray matter was minced, then digested with 1 mg/ml collagenase CLS2 (Worthington) in Dulbecco's modified Eagel's medium (DMEM; Sigma) containing 50 μg/ml gentamycin and 2 mM gultamine in a shaker for 2 h at 37 °C. The cell pallet was separated by centrifugation in 20% bovine serum albumin (BSA)–DMEM (1,000 × g, 20 min). The microvessels obtained in

Results

Electrical resistance across the cell layer was represented in Fig. 4 by Ω/cm2 of the cell layer. Before hypoxia, electrical resistance of the cell layer was significantly lower in endothelial monolayer and pericyte non-contact co-culture (8.0±2.4 and 19.8±2.6 Ω/cm2) than in contact co-culture model (97.5±8.5 Ω/cm2) (n=5, p<0.05). To clarify the role of pericytes in the BBB disruption following ischemia, electrical resistance of the contact co-culture model was compared to that of endothelial

Discussion

The BBB is composed of the microvascular endothelium surrounded by pericytes and astrocyte foot process and is produced by a combination of a specific transport system, a low rate of pinocytes, and the presence of tight junctions that seal adjacent endothelial cells together. It has been proposed that these cells play different roles in the BBB [3]. As a distinctive cell population of the vasculature, pericytes are present in almost all tissues and organ system. While their density and features

Conclusion

In conclusion, we generated a co-culture model using isolated endothelial cells and pericytes, which can evaluate the paracellular and transcellular flux of the BBB in vitro. In this model, pericytes enhanced the inhibition of both paracellular and transcellular flux of the endothelial monolayer in normal conditions. Permeability of the endothelial monolayer and non-contact co-culture was increased following 6 h of hypoxia. Pericyte contact reduced this increased permeability of the endothelial

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