Mechanisms That Regulate Blood-Brain Barrier Dysfunction and Hyperpermeability

Blood-brain barrier (BBB) disruption is the hallmark feature of the secondary injuries that occur following traumatic and ischemic brain injuries. Microvascular hyperpermeability that occurs consequently leads to vasogenic edema and elevated intracranial pressure. Structurally, the BBB is comprised of the brain endothelial cells and the tight junction between them formed by the tight junction proteins (TJPs). Zonula occludens-1 (ZO-1) is an important membrane-bound scaffolding TJP that links transmembrane TJPs to intracellular actin cytoskeletal assembly. Studies indicate that adenosine triphosphate (ATP) that is released into the extracellular space following trauma binds to and activates the purinergic receptor (P2X7R), leading to the production of interleukin-1β (IL-1β); which in turn is shown to activate various downstream proteases. Matrix metalloproteinase-9 (MMP-9) and calpains are proteases known to induce BBB breakdown. However, the mechanisms by which ATP and IL-1β induce BBB dysfunction and hyperpermeability and the involvement of proteases in this process are vastly unknown. We hypothesize that ATP and IL-1β-induced BBB dysfunction and hyperpermeability occurs via P2X7R and /MMP-9/calpain-mediated pathways, respectively. And thus, P2X7R/MMP-9/calpain inhibitions play an important role in mediating BBB dysfunction and hyperpermeability following traumatic injuries in brain. The results from these studies demonstrate that MMP-9/calpain inhibition attenuated IL-1β-induced monolayer hyperpermeability, f-actin stress fiber formation, and loss of ZO-1/TJ integrity and enhanced MMP-9/calpain activity in RBMECs. However, IL-1β treatment did not alter intracellular calcium levels, total number of viable cells, total ZO-1 protein, and mRNA expression. Purinergic receptor (P2X7R) inhibition attenuated Bz-ATP (potent agonist of P2X7R)-induced endothelial cell hyperpermeability, loss of ZO-1/TJ integrity and f-actin stress fiber formation. Although Bz-ATP treatment reduced ZO-1 protein expression, it did not alter total number of viable cells or ZO-1 mRNA expression in vitro. Melatonin (a pineal hormone with MMP-9 inhibitory properties), calpain inhibitor III (calpain inhibitor), and KN-62 (PX7R inhibitor) pretreatment attenuated TBI-induced BBB hyperpermeability in C57BL/6 mice. P2X7R knockout mice demonstrated reduced TBI-induced BBB hyperpermeability compared to the wild-type animals. In conclusion, ATP-sensitive P2X7R are involved in promoting BBB dysfunction and hyperpermeability via IL-1β-mediated activation of MMP-9/calpain pathways and subsequent disorganization of the TJs. Inhibition of this pathway can provide potential therapeutic targets against TBI-induced BBB hyperpermeability in vivo

Synthesis of Neurosteroids: Modulators of NMDA Receptor

Charles University in Prague Faculty of Science Department of Organic and Nuclear Chemistry Karlova Universita v Praze Přírodovědecká fakulta Katedra organické chemie a jaderné chemie Mgr. Eva Šťastná Synthesis of Neurosteroids: Modulators of NMDA receptor Syntéza neurosteroidů: modulátorů NMDA receptoru PhD. Thesis Abstract Autoreferát disetační práce Praha 2009 Prague, 2009 Scientific Presentations and Posters Papers Šťastná E.: Diazomethane (CH2N2). Synlett, 2007,15,2454. Stastna E., Chodounska H., Pouzar V., Kapras V., Borovska J, Cais O., L Vyklicky L.: Synthesis of C3, C5, and C7 pregnane derivatives and their effect on NMDA receptor responses in cultured rat hippocampal neurons. Steroids 2009, 74, 256-263. Kapras V., Šťastná E., Chodounská H., Pouzar V., Krištofíková Z.: Preparation of steroid sulfamates and their interaction with GABAA receptor. Coll. Czech. Chem. Comm., submitted, manuscript number CCCC/2008/000187. Eignerová B., Slavíková B., Buděšínský M., Stastna E., Kotora M.: Synthesis of Fluorinated Brassinosteroids Based on Alkane Cross-Metathesis and Preliminary Biological Assessment. J. Org. Chem., under revision, manuscript number jo-2009- 002079. Patents Stastna E., Chodounska H., Cais O., Vyklicky L., Kapras V., Pouzar V., Kohout L.: Steroidní anionické sloučeniny, způsob..

IL-1β treatment induces dose and time dependent increase in monolayer hyperpermeability.

<p>In Panel A, IL-1β treatment at doses 10, 50 and 100 ng/mL for 2 hours are shown to significantly increase BBB permeability compared to the control group (n = 4; <i>p</i><0.05). Panel B indicates significant increase in IL-1β induced BBB permeability at 2, 3 and 4 hours compared to the control (n = 4; <i>p</i><0.05). Monolayer permeability is expressed as a percentage control of FITC-dextran-10 kDa fluorescent intensity, plotted on the Y-axis. Data are expressed as mean ± % SEM. ‘*a’ indicates significant increase compared to the control group.</p

Melatonin Preserves Blood-Brain Barrier Integrity and Permeability via Matrix Metalloproteinase-9 Inhibition - Fig 9

<p>Melatonin pretreatment attenuates TBI-induced BBB hyperpermeability studied by Evans blue dye extravasation method (Panel A). Pictorial representation of the brain tissue from various groups is shown in Panel 9B. Sham injury group was used as the baseline for all comparisons. Melatonin (10 μg/gram body weight of the animal) pretreatment significantly attenuated TBI-induced Evans blue leakage into the extravascular tissue space (<i>p</i><0.05). Animals were divided into sham (n = 6), vehicle + sham (n = 6), vehicle + TBI (n = 5) and melatonin + TBI (n = 6). Data are expressed as ng/brain cortex ± SEM. ‘*’ indicates statistical significance. ‘a’ indicates significant increase compared to the sham injury/vehicle + sham injury group and ‘b’ indicates significant decrease compared to the vehicle + TBI group.</p

Knockdown of MMP-9 by siRNA attenuates IL-1β treatment-induced monolayer hyperpermeability.

<p>Monolayer permeability is expressed as percentage flux of FITC-dextran-10 kDa fluorescence intensity, plotted on the Y-axis. Data are expressed as mean ± % SEM. ‘*a’ indicates significant increase compared to the control group; ‘*b’ indicates significant decrease compared to the IL-1β (10 ng/mL; 2 hours) treatment group. siRNA transfected groups were compared to control siRNA transfected group (n = 4; p<0.05).</p

MMP-9 inhibitor 1 and melatonin pretreatment attenuates IL-1β treatment- induced MMP-9 activity.

<p>MMP-9 inhibitor 1 (n = 4) and melatonin (n = 5) pretreatment attenuated IL-1β treatment-induced MMP-9 activity in RBMECs. MMP-9 activity is expressed as relative fluorescence units (RFU), plotted on the Y-axis. Data are expressed as mean ± SEM. ‘*a’ indicates significant increase compared to the control group; ‘*b’ indicates significant decrease compared to the IL-1β (10 ng/mL; 2 hours) treatment group. <i>p</i><0.05 was considered statistically significant.</p

IL-1β treatment does not induce cell death.

<p>IL-1β (10 ng/mL; 2 hours) treatment had no effect on cell viability (n = 5). Hydrogen peroxide (used as a positive control) treatment decreases cell viability significantly (<i>p<</i>0.05). Data are expressed as mean ± % SEM. ‘*’ indicates statistical significance. ‘*a’ indicates significant decrease compared to the control group.</p

GM6001, MMP-9 inhibitor 1 and melatonin pretreatment attenuates IL-1β treatment-induced monolayer hyperpermeability.

<p>Panel A indicates the effect of GM6001 (broad-spectrum MMP inhibitor; n = 4); while Panels B and C employ MMP-9 specific inhibitors: MMP-9 inhibitor 1 (n = 4) and melatonin (n = 6) pretreatment on IL-1β (10 ng/mL; 2 hours)—induced monolayer hyperpermeability. Monolayer permeability is expressed as a percentage control of FITC-dextran-10 kDa fluorescence intensity, plotted on the Y-axis. Data are expressed as mean ± % SEM. ‘*a’ indicates significant increase compared to control group; ‘*b’ indicates significant decrease compared to the IL-1β treated group. <i>p</i><0.05 was considered statistically significant.</p

MMP-9 inhibitor 1 and melatonin pretreatment protects against IL-1β treatment-induced loss of ZO-1 junctional integrity.

<p>IL-1β (10 ng/mL; 2 hours) treatment-induced ZO-1 junctional disruption (white arrows) was decreased on pretreatment with MMP-9 inhibitor 1 (n = 4) and melatonin (n = 4).</p