Elsevier

Brain Research

Volume 1189, 16 January 2008, Pages 1-11
Brain Research

Research Report
Pericyte–endothelial cell interaction increases MMP-9 secretion at the blood–brain barrier in vitro

https://doi.org/10.1016/j.brainres.2007.10.099Get rights and content

Abstract

The extracellular matrix (ECM) connecting brain capillary endothelial cells (BCEC) with the surrounding brain resident cells is an essential part of the blood–brain barrier (BBB). Represented by the basement membrane which joins BCEC with brain neuroglia (astrocytes and pericytes) it forms a neurovascular unit. Neuroglia-secreted matrix metalloproteinases (MMPs) control the ECM composition and are involved in the integrity and function of the BBB during cell diapedesis and BBB breakdown after ischemia and other CNS diseases. We examined the involvement of pericytes and astrocytes in endothelial MMP secretion and their effect on endothelial barrier properties in a primary cell culture model using porcine BCEC. We applied puromycin to eliminate pericyte growth and demonstrated a significant (to about 30%) reduction of endothelial MMP-9 production in pericyte-free cultures. In contrast, BCEC co-culture with pericytes resulted in an increased amount of endothelial MMP-9 and active MMPs measured by both zymography and fluorimetric assay. Astrocyte co-culture in a filter setup with BCEC allowing a cell–cell signaling via soluble factors revealed significantly reduced endothelial MMP activity. These data were directly correlated with improved BBB integrity under pericyte elimination and astrocyte co-culture conditions as indicated by transendothelial electrical resistance (TEER) values. Our data define pericyte interactions as a main inducer of endothelial MMP secretion and propose a new role for pericyte–endothelial cell crosstalk at the BBB in vitro.

Introduction

Endothelial cells in the brain microvasculature line the intraluminal portion of brain capillaries and represent the cellular basis of the blood–brain barrier (BBB). These cells are closely interconnected by continuous tight junctions, which prevent the passage of toxic and xenobiotic blood-borne substances into the brain parenchyma (Grant et al., 1998). Although the endothelial cell layer is the principal barrier, the local microenvironment contributes to BBB function. Specialized extracellular matrix (ECM) of the basement membrane (BM) connects endothelial cells with neighboring neuroglial cells like astrocytes and pericytes, forming the so-called neurovascular unit (Hawkins and Davis, 2005). The molecular components of ECM secreted by the surrounding neuroglial cell types and endothelial cells provide an important clue for proper tight junction assembly and BBB properties to sustain function of mature BBB (Arthur et al., 1987, Shivers et al., 1988). During neuroinflammatory conditions the breakdown of the BBB is a key feature associated with an influx of inflammatory cells into the brain. Main mediators of BBB disruption include matrix metalloproteinases (MMPs), which were shown to regulate the structure and function of ECM molecules under normal and pathological conditions (Sternlicht and Werb, 2001, Woessner, 1995). The involvement of MMP-9 in BBB disruption during neuroinflammatory diseases like stroke, multiple sclerosis (MS), and brain injury has been suggested in numerous studies (Anthony et al., 1998, Lukes et al., 1999, Rosenberg, 1995, Rosenberg, 2002, Rosenberg et al., 1994, Rosenberg et al., 1998). The correlation between the expression of different MMPs and the presence of endothelial cells, astrocytes, invading lymphocytes, and pericytes has also been demonstrated (Cottam et al., 1996, Cunningham et al., 2005, Gottschall and Deb, 1996, Hanemaaijer et al., 1993, Harkness et al., 2000). However, the cell type releasing MMP-9 at the normal and pathological BBB has not been identified yet.

The detection of MMP-9 is complicated due to the absence or low secretion of this proteolytic enzyme under steady-state conditions. Although MMP-2 is constitutively expressed and normally present in the brain and cerebrospinal fluid, the expression of MMP-9 can be induced by proinflammatory cytokines (Harkness et al., 2000). During inflammation, infiltrating lymphocytes, endothelial cells, and neuroglia release proinflammatory cytokines which induce the secretion of MMPs (in particular, MMP-9) targeting the BM of the BBB and leading to its disruption (Owens et al., 1994).

Here we studied the effect of MMPs secreted by endothelial cells, pericytes, and astrocytes, on the maintenance of the BBB function in vitro. We used an in vitro model of the blood–brain barrier composed of primary cultures of brain microvascular endothelial cells (Hoheisel et al., 1998, Weidenfeller et al., 2005). For the first time, we present the evidence that a direct pericyte–endothelial cell–cell interaction increases endothelial MMP-9 secretion altering the integrity of the monolayer in vitro. This evidence is important for understanding ECM regulation of BBB properties with respect to the development of future strategies for treatment of pathological conditions of CNS with BBB involvement due to MMP imbalance.

Section snippets

Influence of pericyte presence on endothelial MMP-9 secretion

Baseline expression of endothelial MMP-2 and MMP-9 has been previously demonstrated (Harkness et al., 2000). However, pericytes enclosing the brain microvascular endothelium are difficult to remove from endothelial fraction during capillary isolation (Calabria et al., 2006). In order to clarify whether endothelial cells are the only source of the MMP-9 expression at the BBB, we investigated a pericyte-free monolayer of brain capillary endothelial cells (BCEC). Puromycin, according to the

Discussion

In vivo, astrocytes and pericytes are in close proximity to brain endothelium, and their presence is correlated with the function of the BBB. Both cell types are an important source of basement membrane proteins therefore having great impact on BBB integrity through ECM signaling (Hartmann et al., 2007). The role of matrix metalloproteinases in the brain has been described in several publications (Herron et al., 1986, Rosenberg et al., 2001). Astrocytes, neurons, oligodendrocytes, endothelial

Materials

Preparation media 199 Earle and DME/Ham's F12-medium (1:1) were obtained from Bioconcept (Freiburg, Germany) and supplemented with 0.7 mM l-glutamine, 100 μg/ml gentamicin, 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma) for BCEC culture. DMEM (Dulbecco's modified Eagle's medium) was used for all other cell types. Fetal calf serum (FCS) and 0.25% trypsin solution were purchased from Biochrom (Berlin, Germany). All other chemicals were obtained from Merck (Darmstadt, Germany). The broad

Acknowledgments

The authors are grateful to Sabine Hüwel for the technical assistance. This study was supported by a fellowship awarded to A.Z. by the International Graduate School of Chemistry-Münster (GSC-MS).

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    Present address: Department of Neurology, University of Wuerzburg-Neurology Clinic Josef-Schneider-Straße 11, 97080 Wuerzburg, Germany.

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