Blood-brain barrier dysfunction in ischemic stroke: targeting tight junctions and transporters for vascular protection

Abstract

The blood-brain barrier (BBB) is a physical and biochemical barrier that precisely controls cerebral homeostasis. It also plays a central role in the regulation of blood-to-brain flux of endogenous and exogenous xenobiotics and associated metabolites. This is accomplished by molecular characteristics of brain microvessel endothelial cells such as tight junction protein complexes and functional expression of influx and efflux transporters. One of the pathophysiological features of ischemic stroke is disruption of the BBB, which significantly contributes to development of brain injury and subsequent neurological impairment. Biochemical characteristics of BBB damage include decreased expression and altered organization of tight junction constituent proteins as well as modulation of functional expression of endogenous BBB transporters. Therefore, there is a critical need for development of novel therapeutic strategies that can protect against BBB dysfunction (i.e., vascular protection) in the setting of ischemic stroke. Such strategies include targeting tight junctions to ensure that they maintain their correct structure or targeting transporters to control flux of physiological substrates for protection of endothelial homeostasis. In this review, we will describe the pathophysiological mechanisms in cerebral microvascular endothelial cells that lead to BBB dysfunction following onset of stroke. Additionally, we will utilize this state-of-the-art knowledge to provide insights on novel pharmacological strategies that can be developed to confer BBB protection in the setting of ischemic stroke.

Keywords: blood-brain barrier; endothelial cell; oxidative stress; tight junctions; transporters.

Fig. 1.

Molecular organization of tight junction protein complexes at the mammalian blood-brain barrier. Following onset of ischemic stroke, tight junction protein complexes are disrupted, which leads to breakdown of the blood-brain barrier (BBB). BBB disruption is further exacerbated by degradations of integrins, which are also expressed at the plasma membrane of brain microvascular endothelial cells. ZO-1, zonula occludens-1. [Adapted from Ronaldson and Davis (127) with permission from Bentham Science.]

Fig. 2.

Localization of ATP-binding cassette and solute carrier transporters at the blood-brain barrier. Bcrp, breast cancer resistance protein; Mrp, multidrug resistance protein; Oatp, organic anion transporting polypeptide; OCT, organic cation transporter; P-gp, P-glycoprotein.

Fig. 3.

Mechanisms of blood-brain barrier dysfunction in the setting of ischemic stroke. Early events involved in BBB breakdown following ischemic stroke include stimulation of sodium transporters (i.e., NKCC, NHE), which lead to edema, and degradation of tight junction constituent proteins and integrins, which cause increased paracellular leak at the BBB. BBB breakdown becomes apparent 4–6 h after stroke onset. Later events that contribute to continued barrier disruption include inflammation and accumulation of immune cells (i.e., neutrophils, T-lymphocytes) in brain parenchyma. Overall, BBB breakdown in the setting of ischemic stroke contributes to development of cognitive and motor symptoms. BBB, blood-brain barrier; NHE, Na-H exchanger; NKCC, Na-K-Cl cotransporter; r-tPA, recombinant tissue plasminogen activator.

Fig. 4.

Generation of reactive oxygen species (ROS) in cerebrovascular endothelial cells. During ischemia, mitochondrial superoxide levels increase via nitric oxide (NO) inhibition of cytochrome complexes and oxidation of reducing equivalents in the electron transport chain (ETC). Complex I as well as both sides of complex III (i.e., Q_i and Q_o sites) are the most common sources of mitochondrial superoxide. Superoxide generated within the intermembrane space of mitochondria can reach the cytosol through voltage-dependent mitochondrial anion channels (169). Superoxide levels further increase via cyclooxygenase-2 (COX-2), NADPH oxidase, uncoupled endothelial NO synthase (eNOS), and infiltrating leukocytes. The resulting high levels of superoxide, coupled with the activation of NO-producing eNOS/inducible NOS (iNOS), increases the likelihood of peroxynitrite formation. Peroxynitrite-induced cellular damage includes protein oxidation, tyrosine nitration, DNA damage, and poly(ADP-ribose) polymerase (PARP) activation, lipid peroxidation, and mitochondrial dysfunction. Cyt c, cytochrome c. [Adapted from Thompson and Ronaldson (145) with permission from Elsevier.]

Fig. 5.

Effect of TEMPOL on hypoxia-reoxygenation (H/R)-mediated disruption of the tight junction. Reactive oxygen species (ROS) and subsequent oxidative stress are known to disrupt assembly of critical tight junction proteins such as occludin. Previous data show that administration of TEMPOL, a ROS scavenger, prevents disruption of occludin oligomers. Furthermore, TEMPOL attenuates the increase in sucrose leak across the blood-brain barrier observed in animals subjected to H/R stress. These observations demonstrate that the tight junction can be targeted pharmacologically in an effort to confer vascular protection during ischemic stroke. Nx, normoxia; S, sucrose; T, TEMPOL. [Adapted from Ronaldson and Davis (127) with permission from Bentham Science.]

Fig. 6.

Prevention of blood-brain barrier (BBB) dysfunction by targeting multidrug resistance protein (Mrp) isoforms in brain microvessel endothelial cells. Our laboratory has reported that expression of Mrp1, Mrp2, and Mrp4 are increased at the BBB in the setting of hypoxia/reoxygenation (H/R) stress. H/R stress is known to decrease glutathione (GSH) levels and increase glutathione disulfide (GSSG) concentrations in brain microvascular endothelial cells. We postulate that changes in GSH/GSSG transport occur during H/R as a result of altered functional expression of at least one Mrp isoform. Since nuclear factor E2-related factor-2 (Nrf2), a ROS sensitive transcription factor, is known to regulate Mrps, we propose that this signaling pathway is a central mechanism for regulation of Mrps at the BBB. Antioxidant drugs can be used as pharmacological tools to understand how targeting activation of the Nrf2 pathway can control Mrp expression and/or activity.