Exogenous expression of claudin‐5 induces barrier properties in cultured rat brain capillary endothelial cells

Abstract

Claudins are thought to be major components of tight junctions (TJs), and claudin-5 and -12 are localized at TJs of the blood-brain barrier (BBB). Claudin-5-deficient mice exhibit size-selective (<800 Da) opening of the BBB. The purpose of this study was to clarify the expression levels of claudin-5 and -12 in rat brain capillary endothelial cells, and to examine the ability of claudin-5 to form TJs in cultured rat brain capillary endothelial cells (TR-BBB). Expression of claudin-5 mRNA in rat brain capillary fraction was 751-fold greater than that of claudin-12. The level of claudin-5 mRNA in the rat brain capillary fraction (per total mRNA) was 35.6-fold greater than that in whole brain, while the level of claudin-12 mRNA was only 13.9% of that in whole brain, suggesting that expression of claudin-12 mRNA is not restricted to brain capillaries. Transfection of TR-BBB cells with the claudin-5 gene afforded TR-BBB/CLD5 cells, which showed no change in expression of claudin-12 or ZO-1, while the expressed claudin-5 was detected at the cell-cell boundaries. The permeability surface product of [ 14 C]inulin at a TR-BBB/CLD5 cell monolayer was significantly smaller (P < 0.01) than that for the parental TR-BBB cells, and the values of the permeability coefficient (Pe) were 1.14 Â 10 À3 and 11.6 Â 10 À3 cm/ min, respectively. These results indicate that claudin-5, but not claudin-12, is predominantly expressed in brain capillaries, and plays a key role in the appearance of barrier properties of brain capillary endothelial cells. J. Cell. Physiol. 210: 81-86, 2007. Tight junctions (TJs) between brain capillary endothelial cells play a key role in the formation of the blood-brain barrier (BBB) because TJs block paracellular diffusion between endothelial cells (Pardridge, 2005). To date, three distinct types of integral membrane proteins have been reported to be localized at TJs: occludin (Furuse et al., 1993), junctional adhesion molecule (JAM) (Martin-Padura et al., 1998), and claudins (Furuse et al., 1998a). Among these TJ proteins, claudins are thought to be the major constituents of TJs (Furuse et al., 1998b). Claudins are a multigene family consisting of more than 20 members (Tsukita et al., 2001). They interact homogeneously and heterogeneously with each other to form TJs, and the properties of the resulting TJs depend on the claudin subtypes involved . Therefore, clarifying the contributions of claudin subtypes to TJ formation in the BBB is important in order to understand the development of the BBB and changes in the BBB that may occur in disease, as well as for developing novel techniques to deliver drugs to the brain.Claudin-5 is localized at the cell-cell boundaries of brain capillary endothelial cells (Morita et al., 1999). Nitta et al. (2003) reported size-selective opening of the BBB in claudin-5-deficient mice. In these mice, TJs with a normal appearance were present at the brain capillary endothelial cell-cell contact regions, but leakage of small molecules (<800 Da), though not larger molecules, was obs...

VEGF-mediated disruption of endothelial CLN-5 promotes blood-brain barrier breakdown

“…Although CLN-3, CLN-5, and CLN-12 are all expressed by BMVECs (4), compelling evidence implicates CLN-5 as primarily responsible for determining the high trans-endothelial resistance of BBB tight junctions (5,6). Within the brain, CLN-5 is restricted to endothelium and expression of its mRNA within this compartment is more than 750 times that of CLN-12 (5).…”

“…Claudins act as the main determinants of tight junction properties (3), and BMVECs predominantly express CLN-5, with smaller amounts of CLN-3 and CLN-12 (4). Transfection studies show that CLN-5 plays a key role in the appearance of BBB properties (5), and CLN-5 -/-mice display selective BBB opening (6). OCLN is also a tight junction strand component, and contributes to junction properties and regulates permeability (7), but strands form normally in OCLN-deficient cells (8), and OCLN -/-mice display a complex phenotype (9).…”

Roles of the Cyclooxygenase 2 Matrix Metalloproteinase 1 Pathway in Brain Metastasis of Breast Cancer

“…The tight junction of BBB consists of transmembrane molecules, Occludin, Claudin, and junctional adhesion proteins that are linked to the actin cytoskeleton through cytoplasmic accessory proteins including Zo-1, Zo-2, and Zo-3 (5,6). Occludin and Claudin were identified particularly as critical proteins of BBB integrity (37)(38)(39)(40)(41). In this study we have shown A, tumor initiating cells were prepared from MB-MB231 and 231BrM using CD24, CD44, and ESA markers as described under "Experimental Procedures."…”

Interendothelial Claudin-5 Expression Depends on Cerebral Endothelial Cell–Matrix Adhesion by β1-Integrins

“…It resides within cerebral and pulmonary endothelial cells at the cell-cell border, but not in epithelial cells, including astrocyte end-feet (Nitta et al, 2003). Claudin-5 is predominantly expressed in brain capillaries, and appears to have a key role in the barrier to egress of small molecules by brain capillary endothelial cells (Morita et al, 1999;Ohtsuki et al, 2007). In one study, the normalized claudin-5 mRNA level in the rat brain capillary fraction was 35.6-fold greater than that in the brain (Ohtsuki et al, 2007).…”

“…After 7 days hypoxia, retinal vascular claudin-5 expression in the rat decreases and extravasation of an injected 534 Da tracer increases compared with normoxic retina (Koto et al, 2007). Complementary work in culture has demonstrated that after transfection of claudin-5 gene into rat brain capillary endothelial cells, the inulin paracellular permeability coefficient of the endothelial cell monolayer decreases (Ohtsuki et al, 2007). Separately, increased claudin-5 protein expression paralleled an increase in the transendothelial electrical resistance (TEER) of bEnd.3 monolayers during the growth phase.…”