Research ReportPericyte–endothelial cell interaction increases MMP-9 secretion at the blood–brain barrier 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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- 1
Present address: Department of Neurology, University of Wuerzburg-Neurology Clinic Josef-Schneider-Straße 11, 97080 Wuerzburg, Germany.
- 2
Both authors have contributed equally to this study.