Interendothelial Tight Junctions Research Articles

Matrix Metalloproteinase-9 inhibitors as therapeutic drugs for traumatic brain injury

Traumatic brain injury (TBI) is one of the leading causes of morbidity and mortality among young adults and the elderly. In the United States, TBI is responsible for around 30 percent of all injuries brought on by injuries in general. Vasogenic cerebral edema due to blood-brain barrier (BBB) dysfunction and the associated elevation of intracranial pressure (ICP) are some of the major causes of secondary injuries following traumatic brain injury. Matrix metalloproteinase-9 (MMP-9) is a therapeutic target for being an enzyme that degrades the proteins that make up a part of the microvascular basal lamina as well as inter-endothelial tight junctions of the blood-brain barrier. MMP-9-mediated BBB dysfunctions and the compromise of the BBB is a major pathway that leads the development of vasogenic cerebral edema, elevation of ICP, poor cerebral perfusion and brain herniation following traumatic brain injury. That makes MMP-9 an effective therapeutic target and endogenous or exogenous MMP-9 inhibitors as therapeutic drugs for preventing secondary brain damage after traumatic brain injury. Although our understanding of the mechanisms that underlie the primary and secondary stages of damage following a TBI has significantly improved in recent years, such information has not yet resulted in the successful development of novel pharmacological treatment options for traumatic brain injury. Recent pre-clinical and/or clinical studies have demonstrated that there are several compounds with specific or non-specific MMP-9 inhibitory properties either directly binding and inhibiting MMP-9 or by indirectly inhibiting MMP-9, with potential as therapeutic agents for traumatic brain injury. This article reviews the efficacy of several such medications and potential agents that include endogenous and exogeneous compounds that are at various levels of research and development. MMP-9-based therapeutic drug development has enormous potential in the pharmacological treatment of cerebral edema and/or neuronal injury resulting from traumatic brain injury.

Determination of Tight Junction Integrity in Brain Endothelial Cells Based on Tight Junction Protein Expression.

Blood-brain barrier (BBB) dysfunction and hyperpermeability that occurs following traumatic and ischemic insults lead to various downstream ill effects such as cerebral edema and elevation of intracranial pressure. The inter-endothelial tight junctions that consist of tight junction proteins are critical regulators of BBB dysfunctions and hyperpermeability. The major tight junction-associated proteins of the BBB are occludin, claudins, and junctional adhesion molecules that are intracellularly linked to the adaptor protein zonula occludens-1 (ZO-1). Quantitative measurement of tight junction-associated proteins provides valuable insight into barrier integrity and mechanisms that regulate microvascular hyperpermeability. Western blot analysis is a commonly used method to separate and identify proteins in a mixture using gel electrophoresis. Understanding the changes in the expression of one or more of these proteins is critical to evaluating barrier integrity and permeability in health and disease. Furthermore, studying them will provide insight into the associated downstream signaling pathways and evaluation of therapeutic approaches for regulating BBB permeability. Herein, we have described the protocol for immunoblot analysis of ZO-1 as an indicator of tight junction integrity in brain microvascular endothelial cells.

Andrographolide‐Mediated Protection of Barrier Integrity and Permeability is Mediated Through NLRP3 Inflammasome Inhibition in Blood‐Brain Barrier Endothelial Cells

The blood‐brain barrier (BBB) that consists of the inter‐endothelial tight junctions, acts as the major blood‐brain interface and regulator of brain microvascular hyperpermeability. In the brain, the loss of BBB integrity and hyperpermeability leads to cerebral edema formation and intracranial pressure elevation in traumatic brain injury and ischemic strokes. Recent studies demonstrated that reactive oxygen species are potential signals for the NLR family pyrin domain containing 3 (NLRP3) inflammasome activation that leads to interleukin‐1β (IL‐1β) secretion. IL‐1β plays a major role in promoting BBB hyperpermeability in various brain pathologies, including TBI. Furthermore, recent studies from our lab have demonstrated a tri‐phasic role for hydrogen peroxide (H2O2) in BBB endothelial cells where it promotes, angiogenesis, hyperpermeability and apoptosis in a tri‐phasic and concentration dependent manner. Hydrogen peroxide plays a major role physiologically as the second messenger and pathologically as an inducer of oxidative stress and associated cellular signaling. We hypothesized that in BBB endothelial cells, H2O2‐induced barrier dysfunction and hyperpermeability are NLRP3 inflammasome‐dependent. The main objective of our study was to evaluate the role of NLRP3 inflammasome signaling in H2O2‐mediated barrier dysfunction and hyperpermeability in human brain microvascular endothelial cells and to test whether andrographolide, a labdane diterpenoid possessing potent anti‐inflammatory properties will attenuate such effects. Th effect of H2O2 treatment on tight junction integrity/permeability was studied using zonula occludens‐1 (ZO‐1) immunofluorescence localization, ZO‐1 immunoblot analysis, Transwell monolayer permeability assay using FITC‐dextran (10‐kDa) as a fluorescent marker and cell viability/apoptosis assay. The effect of NLRP3 inhibitor (MCC950) and andrographolide on H2O2‐induced hyperpermeability was studied using Transwell permeability assay. Our results show that, CRISPR/Cas‐9‐mediated knockdown of ZO‐1 resulted in monolayer hyperpermeability demonstrating the significance of ZO‐1 in regulating barrier functions. CRISPR‐based activation of NLRP3 induced monolayer hyperpermeability whereas CRISPR/Cas‐9‐based NLRP3 knockdown had no significant effect on permeability. H2O2 (10µM) induced monolayer hyperpermeability and the effect was decreased by MCC950 and andrographolide treatment. Hydrogen peroxide induced cathepsin B (an activator of the NLRP3 pathway) activity significantly, and the effect was decreased by andrographolide treatment. H2O2‐mediated barrier dysfunction and hyperpermeability were not due to changes in cell viability/apoptosis. These results suggest that NLRP3 is a mediator of H2O2‐induced barrier dysfunction and hyperpermeability in human BBB endothelial cells and andrographolide is an effective inhibitor of this pathway.

Overcoming Blood-Brain Barrier Resistance: Implications for Extracellular Vesicle-Mediated Drug Brain Delivery

Drug delivery across the blood–brain barrier (BBB) has several challenges, especially toward targeting neurological diseases, due to tight and selective barrier function of the BBB. Several structural and functional components of this barrier contribute to restricting drug entry, such as interendothelial tight junctions (TJs), efflux transporters, drug-metabolizing enzymes, and crosstalk between the cells of the neurovascular unit. Among different strategies to overcome BBB resistance to therapeutic drug delivery, the use of extracellular vesicles (EVs) gained attention in recent years. This review discusses the BBB structural and functional resistance, as well as potential avenues to overcome this challenge using EVs as drug delivery vehicles into the brain.

LECT2 Ameliorates Blood–Retinal Barrier Impairment Secondary to Diabetes Via Activation of the Tie2/Akt/mTOR Signaling Pathway

PurposeCurrent treatments for diabetic retinopathy (DR) have considerable limitations, emphasizing the need for new therapeutic options. The effect of leukocyte cell-derived chemotaxin 2 (LECT2) on diabetes-induced blood–retinal barrier impairment and the possible underlying mechanism were investigated both in vivo and in vitro.MethodsTwenty diabetic and 22 nondiabetic eyes were included in this study. Additionally, we established a streptozotocin-induced diabetic mouse model and observed vascular leakage in mice treated with or without recombinant LECT2 (rLECT2) intravitreal injection (40 µg/mL, 1 µL). The levels of LECT2 and interendothelial junction proteins (ZO1, VE-cadherin, and occludin) were analyzed by western blot and/or immunofluorescence. Endothelial junctions in mouse retinas were observed by transmission electron microscopy (TEM). Moreover, confluent human retinal microvascular endothelial cells (HRMECs) and human umbilical vein endothelial cells (HUVECs) were treated (0–72 hours) with glucose (0 or 30 mM) in the presence or absence of rLECT2 (40–360 ng/mL). After treatment, intact cell monolayers were monitored for permeability to 40-kD FITC-dextran. Interendothelial junction targets and Tie2/Akt/mTOR signaling pathway components were investigated by western blot.ResultsIn diabetic human and mouse retinas and high-glucose (30 mM)–treated HRMECs and HUVECs, the levels of LECT2 and interendothelial junction proteins were decreased. rLECT2 treatment (80 ng/mL) significantly attenuated the hyperglycemia-induced reduction in endothelial cell barrier function and inhibited the migration and tube formation of HRMECs and HUVECs. In addition, rLECT2 increased the levels of interendothelial junction proteins via activation of the Tie2/Akt/mTOR signaling pathway. Furthermore, intravitreal rLECT2 injections increased the levels of interendothelial junction proteins and reversed diabetes-induced junction disruption.ConclusionsrLECT2 can increase the levels of interendothelial tight junction proteins through activation of the Tie2/Akt/mTOR signaling pathway and can ameliorate inner blood–retinal barrier impairment secondary to diabetes. LECT2 might be a potential target to prevent the progression of DR.

Fluorescein Permeability of the Blood-Brain Barrier Is Enhanced in Juvenile- but Not Young Adult-Onset Type 1 Diabetes in Rats.

Clinically, neurological disorders, such as cognitive impairments and dementia, have been reported as diabetic complications, which are remarkable, especially in children with diabetes. The blood-brain barrier (BBB) is a physiologically dynamic regulatory barrier that maintains the consistency of the fluid microenvironment composition of the brain. However, the differences in BBB conditions between children and adults and the contribution of the BBB to the severity of cognitive impairments remain unclear. We generated adult-onset diabetes mellitus (DM) and juvenile-onset diabetes mellitus (JDM) diabetic rat models and investigated BBB functions in these models during the early stages of type 1 diabetes. We performed a BBB permeability assay using sodium fluorescein, a small-molecule fluorescent dye, to evaluate endothelial transport from the blood to the central nervous system. One week after diabetes onset, BBB permeability increased in the hippocampus and striatum of JDM rats, but no changes were observed in the frontal cortex and hypothalamus of JDM rats or for any region of DM rats. The double staining of tight junction proteins and astrocytes revealed no changes in the hippocampus and striatum of JDM rats. These results suggested that the observed increase in BBB permeability during early-stage diabetes onset in JDM rats, which did not depend on the expression of the interendothelial tight junction protein, claudin-5, may affect stylized neural development and cognitive function.

Lipopolysaccharide influences the plasma and brain pharmacokinetics of subcutaneously-administered HsTX1[R14A], a KV1.3-blocking peptide

KV1.3 is a voltage-gated potassium channel that is upregulated in neuroinflammatory conditions, such as Alzheimer's disease and Parkinson's disease. HsTX1[R14A] is a potent and selective peptide blocker of KV1.3 with the potential to block microglial KV1.3, but its brain uptake is expected to be limited owing to the restrictive nature of the blood-brain barrier. To assess its peripheral and brain exposure, a LC-MS/MS assay was developed to quantify HsTX1[R14A] concentrations in mouse plasma and brain homogenate that was reliable and reproducible in the range of 6.7–66.7 nM (r2 = 0.9765) and 15–150 pmol/g (r2 = 0.9984), respectively. To assess if neuroinflammation affected HsTX1[R14A] disposition, C57BL/6 mice were administered HsTX1[R14A] subcutaneously (2 mg/kg) 24 h after an intraperitoneal dose of Escherichia coli lipopolysaccharide (LPS), which is commonly used to induce neuroinflammation; brain and plasma concentrations of HsTX1[R14A] were then quantified over 120 min. LPS treatment significantly retarded the decline in HsTX1[R14A] plasma concentrations, presumably as a result of reducing renal clearance, and led to substantial brain uptake of HsTX1[R14A], presumably through disruption of brain inter-endothelial tight junctions. This study suggests that HsTX1[R14A] may reach microglia in sufficient concentrations to block KV1.3 in neuroinflammatory conditions, and therefore has the potential to reduce neurodegenerative diseases.

Blood-brain barrier permeability towards small and large tracers in a mouse model of osmotic demyelination syndrome

During osmotic demyelination syndrome (ODS), myelin and oligodendrocyte are lost according to specific patterns in centro- or extra-pontine regions. In both experimental model of ODS and human cases, brain lesions are locally correlated with the disruption of the blood brain-barrier (BBB). The initiation, the degree and the duration of blood-brain barrier (BBB) opening as well as its contribution to brain damages are still a matter of debate. Using a panel of intravascular tracers from low- to high- molecular weight (from 0.45 kDa 150 kDa), we have assessed the BBB permeability at different timings of ODS induced experimentally in mice. ODS was mimicked according to a protocol of rapid correction of a chronic hyponatremia. We demonstrated that BBB leakage towards smallest tracers Lucifer Yellow (0.45 kDa) and Texas Red-dextran (3 kDa) was delayed by 36 h compared to the first clues of oligodendrocyte loss (occurring 12 h post-correction of hyponatremia). At 48 h post-correction and concomitantly to myelin loss, BBB was massively disrupted as attested by accumulation of Evans Blue (69 kDa) and IgG (150 kDa) in brain parenchyma. Analysis of BBB ultrastructure verified that brain endothelial cells had minimal alterations during chronic hyponatremia and at 12 h post-correction of hyponatremia. However, brain endothelium yielded worsened alterations at 48 h, such as enlarged vesicular to tubular-like cytoplasmic profiles of pinocytosis and/or transcytosis, local basal laminae abnormalities and sub-endothelial cavities. The protein expressions of occludin and claudin-1, involved in inter-endothelial tight junctions, were also downregulated at 48 h post-correction of hyponatremia. Our results revealed that functional BBB opening occured late in pre-established ODS lesions, and therefore was not a primary event initiating oligodendrocyte damages in the mouse model of ODS.

Sleep loss disrupts pericyte-brain endothelial cell interactions impairing blood-brain barrier function

Sleep loss in the rat increases blood-brain barrier permeability to circulating molecules by disrupting interendothelial tight junctions. Despite the description of the ultrastructure of cerebral microvessels and the evidence of an apparent pericyte detachment from capillary wall in sleep restricted rats the effect of sleep loss on pericytes is unknown. Here we characterized the interactions between pericytes and brain endothelial cells after sleep loss using male Wistar rats. Animals were sleep-restricted 20 h daily with 4 h sleep recovery for 10 days. At the end of the sleep restriction, brain microvessels (MVs) were isolated from cerebral cortex and hippocampus and processed for Western blot and immunocytochemistry to evaluate markers of pericyte-endothelial cell interaction (connexin 43, PDGFR-β), tight junction proteins, and proinflammatory mediator proteins (MMP9, A2A adenosine receptor, CD73, NFκB). Sleep restriction reduced PDGFR-β and connexin 43 expression in MVs; in addition, scanning electron microscopy micrographs showed that pericytes were detached from capillary walls, but did not undergo apoptosis (as depicted by a reduced active caspase-3 expression). Sleep restriction also decreased tight junction protein expression in MVs and increased BBB permeability to low- and high-molecular weight tracers in in vivo permeability assays. Those alterations seemed to depend on a low-grade inflammatory status as reflected by the increased expression of phosphorylated NFκB and A2A adenosine receptor in brain endothelial cells from the sleep-restricted rats. Our data show that pericyte-brain endothelial cell interaction is altered by sleep restriction; this evidence is essential to understand the role of sleep in regulating blood-brain barrier function.

Contribution of cathepsin B-dependent Nlrp3 inflammasome activation to nicotine-induced endothelial barrier dysfunction

Recent studies indicate that endothelial Nlrp3 inflammasome is critically involved in the development of cardiovascular complications. However, it remains unknown whether endothelial inflammasome is involved in endothelial barrier dysfunction associated with smoking. This study aims to investigate the role of endothelial Nlrp3 inflammasome in nicotine-induced disruption of inter-endothelial tight junctions and consequent endothelial barrier dysfunction. The confocal microscopic analysis demonstrated that mice treated with nicotine exhibited disrupted inter-endothelial tight junctions as shown by decreased ZO-1 and ZO-2 expression in the coronary arterial endothelium, whereas the decreases in ZO-1/2 were prevented by Nlrp3 gene deficiency. In cultured endothelial cells, nicotine caused Nlrp3 inflammasome complex formation and enhances the inflammasome activity as shown by increased cleavage of pro-caspase-1, and interleukin-1β (IL-1β) production. Further, nicotine disrupted tight junction and increased permeability in an endothelial cell monolayer, and this nicotine-induced effect was prevented by silencing of Nlrp3 gene, inhibition of caspase-1, or blockade of high mobility group box 1 (HMGB1). Nicotine increased endothelial cell lysosomal membrane permeability and triggered the lysosomal release of cathepsin B, whereas these events were prevented by pretreating cells with a lysosome stabilizing agent, dexamethasone. Collectively, our data suggest that nicotine enhances cathepsin B-dependent Nlrp3 inflammasome activation and the consequent production of a novel permeability factor HMGB1, which causes disruption of inter-endothelial tight junctions leading to endothelial hyperpermeability. Instigation of endothelial inflammasomes may serve as an important pathogenic mechanism contributing to the early onset of vasculopathy associated with smoking.

Molecular Ultrasound Imaging of Junctional Adhesion Molecule A Depicts Acute Alterations in Blood Flow and Early Endothelial Dysregulation.

The junctional adhesion molecule A (JAM-A) is physiologically located in interendothelial tight junctions and focally redistributes to the luminal surface of blood vessels under abnormal shear and flow conditions accompanying atherosclerotic lesion development. Therefore, JAM-A was evaluated as a target for molecularly targeted ultrasound imaging of transient endothelial dysfunction under acute blood flow variations. Flow-dependent endothelial dysfunction was induced in apolipoprotein E-deficient mice (n=43) by carotid partial ligation. JAM-A expression was investigated by molecular ultrasound using antibody-targeted poly(n-butyl cyanoacrylate) microbubbles and validated with immunofluorescence. Flow disturbance and arterial remodeling were assessed using functional ultrasound. Partial ligation led to an immediate drop in perfusion at the ligated side and a direct compensatory increase at the contralateral side. This was accompanied by a strongly increased JAM-A expression and JAM-A-targeted microbubbles binding at the partially ligated side and by a moderate and temporary increase in the contralateral artery (≈14× [P<0.001] and ≈5× [P<0.001] higher than control, respectively), both peaking after 2 weeks. Subsequently, although JAM-A expression and JAM-A-targeted microbubbles binding persisted at a higher level at the partially ligated side, it completely normalized within 4 weeks at the contralateral side. Temporary blood flow variations induce endothelial rearrangement of JAM-A, which can be visualized using JAM-A-targeted microbubbles. Thus, JAM-A may be considered as a marker of acute endothelial activation and dysfunction. Its imaging may facilitate the early detection of cardiovascular risk areas, and it enables the therapeutic prevention of their progression toward an irreversible pathological state.

Blood-Brain Barrier Changes in High Altitude.

Cerebral syndromes related to high-altitude exposure are becoming more frequent as the number of trips to high altitudes has increased in the last decade. The commonest symptom is headache, followed by acute mountain sickness (AMS) and high-altitude cerebral edema (HACE), which can be fatal. The pathophysiology of these syndromes is not fully understood. The classical "tight-fit hypothesis" posits that there are some anatomical variations that would obstruct the sinovenous outflow and worsen vasogenic edema and intracranial hypertension reactive to hypoxia. This could explain microhemorrhages seen in autopsies. However, recent magnetic resonance imaging studies have demonstrated some components of cytotoxic edema in HACE absent in AMS, suggesting a dysfunction in water balance at the cellular level. Currently, the "red-ox theory" supports trigemino-vascular system activation by free radicals formed after hypoxia and the consequent oxidative stress cascades. Apart from trigemino-vascular system activation, free radicals can also provoke membrane destabilisation mediated by lipid peroxidation, inflammation, and local hypoxia inducible factor-1α and vascular endothelial growth factor activation, resulting in gross blood-brain barrier (BBB) dysfunction. Besides alterations in endothelial cells such as increased pinocytotic vesicles and disassembly of interendothelial tight junction proteins, capillary permeability may also increase with subsequent swelling of astrocyte end-feet. In conclusion, although the pathophysiology of AMS and HACE is not completely understood, recent evidence proposes a multifactorial entity, with brain swelling and compromise of the BBB considered to play an important role. A fuller comprehension of these processes is crucial to reduce and prevent BBB alterations during high-altitude exposure.

Enhancement of endothelial permeability by free fatty acid through lysosomal cathepsin B-mediated Nlrp3 inflammasome activation.

Obesity is an important risk factor for exacerbating chronic diseases such as cardiovascular disease and cancer. High serum level of saturated free fatty acids such as palmitate is an important contributor for obesity-induced diseases. Here, we examined the contribution of inflammasome activation in vascular cells to free fatty acid-induced endothelial dysfunction and vascular injury in obesity. Our findings demonstrated that high fat diet-induced impairment of vascular integrity and enhanced vascular permeability in the myocardium in mice were significantly attenuated by Nlrp3 gene deletion. In microvascular endothelial cells (MVECs), palmitate markedly induces Nlrp3 inflammasome complex formation leading to caspase-1 activation and IL1β production. By fluorescence microscopy and flow cytometry, we observed that such palmitate-induced Nlrp3 inflammasome activated was accompanied by a reduction in inter-endothelial tight junction proteins ZO-1/ZO-2. Such palmitate-induced decrease of ZO-1/ZO-2 was also correlated with an increase in the permeability of endothelial monolayers treated with palmitates. Moreover, palmitate-induced alterations in ZO-1/ZO-2 or permeability were significantly reversed by an inflammasome activity inhibitor, YVAD, or a high mobility group box 1 (HMGB1) activity inhibitor glycyrrhizin. Lastly, blockade of cathepsin B with Ca-074Me significantly abolished palmitate-induced activation of Nlrp3 inflammasomes, down-regulation of ZO-1/ZO-2, and enhanced permeability in MVECs or their monolayers. Together, these data strongly suggest that activation of endothelial inflammasomes due to increased free fatty acids produces HMGB1, which disrupts inter-endothelial junctions and increases paracellular permeability of endothelium contributing to early onset of endothelial injury during obesity.

Research on the protective effect of diazoxide pretreatment on the blood-brain barrier of rats after cerebral ischemia/reperfusion injury

Objective To investigate the effect and its mechanism of diazoxide on the blood-brain barrier (BBB) of rats after cerebral ischemia/reperfusion (I/R) injury. Methods Sixty Wistar rats were randomly divided into sham operation group, I/R group, and diazoxide pretreatment groups of low, middle, large dose (5, 10, 20 mg/kg). The I/R models of rats were performed to undergo middle cerebral artery embolism by thread. BBB permeability was estimated by Evans blue (EB) dyeing, transmission electron microscope (TEM) was used to observe the modification of interendothelial tight junction (TJ) of capillaries. The expression of aquaporin-4 (AQP4) in every rat brain tissues was detected by immunity histochemistry technique. Results ⑴ Compared to sham operation group, the permeability extent of EB were significantly increased by I/R, which was distinctly attenuated in middle and large dose of diazoxide pretreatment rats, while no obvious changes were found between I/R and low dose groups. ⑵ TEM showed that TJ of the brain tissue opened after I/R injury and no significant opening of TJ was observed in middle and large dose of diazoxide preconditioning groups. ⑶ Compared to sham operation group, the expression of AQP4 in the brain tissue of the I/R group was apparently increased (P<0.01). Compared to I/R group, the expression of AQP4 was apparently increased in middle and large dose pretreatment groups (P<0.01), and there were no obvious difference between low dose group and the I/R group. Conclusions Preconditioning of ischemia/reperfusion injury with diazoxide protects the blood-brain barrier, which may due to keep the TJ closed and decrease expression of AQP4 protein. Key words: Diazoxide/PD; Ischemic preconditioning; Brain ischemia; Reperfusion injury/DT; Blood-brain barrier/DE

High-Field MRI Reveals a Drastic Increase of Hypoxia-Induced Microhemorrhages upon Tissue Reoxygenation in the Mouse Brain with Strong Predominance in the Olfactory Bulb.

Human pathophysiology of high altitude hypoxic brain injury is not well understood and research on the underlying mechanisms is hampered by the lack of well-characterized animal models. In this study, we explored the evolution of brain injury by magnetic resonance imaging (MRI) and histological methods in mice exposed to normobaric hypoxia at 8% oxygen for 48 hours followed by rapid reoxygenation and incubation for further 24 h under normoxic conditions. T2*-, diffusion-weighted and T2-relaxometry MRI was performed before exposure, immediately after 48 hours of hypoxia and 24 hours after reoxygenation. Cerebral microhemorrhages, previously described in humans suffering from severe high altitude cerebral edema, were also detected in mice upon hypoxia-reoxygenation with a strong region-specific clustering in the olfactory bulb, and to a lesser extent, in the basal ganglia and cerebral white matter. The number of microhemorrhages determined immediately after hypoxia was low, but strongly increased 24 hours upon onset of reoxygenation. Histologically verified microhemorrhages were exclusively located around cerebral microvessels with disrupted interendothelial tight junction protein ZO-1. In contrast, quantitative T2 and apparent-diffusion-coefficient values immediately after hypoxia and after 24 hours of reoxygenation did not show any region-specific alteration, consistent with subtle multifocal but not with regional or global brain edema.

The blood-brain barrier and methamphetamine: open sesame?

The chemical and electrical microenvironment of neurons within the central nervous system is protected and segregated from the circulation by the vascular blood–brain barrier. This barrier operates on the level of endothelial cells and includes regulatory crosstalk with neighboring pericytes, astrocytes, and neurons. Within this neurovascular unit, the endothelial cells form a formidable, highly regulated barrier through the presence of inter-endothelial tight junctions, the absence of fenestrations, and the almost complete absence of fluid-phase transcytosis. The potent psychostimulant drug methamphetamine transiently opens the vascular blood–brain barrier through either or both the modulation of inter-endothelial junctions and the induction of fluid-phase transcytosis. Direct action of methamphetamine on the vascular endothelium induces acute opening of the blood-brain barrier. In addition, striatal effects of methamphetamine and resultant neuroinflammatory signaling can indirectly lead to chronic dysfunction of the blood-brain barrier. Breakdown of the blood-brain barrier may exacerbate the neuronal damage that occurs during methamphetamine abuse. However, this process also constitutes a rare example of agonist-induced breakdown of the blood-brain barrier and the adjunctive use of methamphetamine may present an opportunity to enhance delivery of chemotherapeutic agents to the underlying neural tissue.

Matrix metalloproteinase-9 mediates post-hypoxic vascular pruning of cerebral blood vessels by degrading laminin and claudin-5.

Vascular remodeling involves a highly coordinated break-down and build-up of the vascular basal lamina and inter-endothelial tight junction proteins. In light of the important role of matrix metalloproteinases (MMPs) in tissue remodeling, the goal of this study was to examine the role of MMP-9 in remodeling of cerebral blood vessels, both in hypoxia-induced angiogenesis and in the vascular pruning that accompanies the switch from hypoxia back to normoxia. In a chronic mild hypoxia model of cerebrovascular remodeling, gel zymography revealed that MMP-9 levels were increased, both during hypoxic-induced angiogenesis and in the post-hypoxic pruning response. Interestingly, compared to wild-type mice, MMP-9 KO mice showed no alteration in hypoxic-induced angiogenesis, but did show marked delay in post-hypoxic vascular pruning. In wild-type mice, vascular pruning was associated with fragmentation of vascular laminin and the tight junction protein claudin-5, while this process was markedly attenuated in MMP-9 KO mice. In vitro experiments showed that hypoxia stimulated MMP-9 expression in brain endothelial cells but not pericytes. These results show that while MMP-9 is not essential for hypoxic-induced cerebral angiogenesis, it plays an important role in post-hypoxic vascular pruning by degrading laminin and claudin-5.

Dimethyl fumarate attenuates cerebral edema formation by protecting the blood–brain barrier integrity

Brain edema is a hallmark of various neuropathologies, but the underlying mechanisms are poorly understood. We aim to characterize how tissue hypoxia, together with oxidative stress and inflammation, leads to capillary dysfunction and breakdown of the blood–brain barrier (BBB). In a mouse stroke model we show that systemic treatment with dimethyl fumarate (DMF), an antioxidant drug clinically used for psoriasis and multiple sclerosis, significantly prevented edema formation in vivo. Indeed, DMF stabilized the BBB by preventing disruption of interendothelial tight junctions and gap formation, and decreased matrix metalloproteinase activity in brain tissue. In vitro, DMF directly sustained endothelial tight junctions, inhibited inflammatory cytokine expression, and attenuated leukocyte transmigration. We also demonstrate that these effects are mediated via activation of the redox sensitive transcription factor NF-E2 related factor 2 (Nrf2). DMF activated the Nrf2 pathway as shown by up-regulation of several Nrf2 target genes in the brain in vivo, as well as in cerebral endothelial cells and astrocytes in vitro, where DMF also increased protein abundance of nuclear Nrf2. Finally, Nrf2 knockdown in endothelial cells aggravated subcellular delocalization of tight junction proteins during ischemic conditions, and attenuated the protective effect exerted by DMF. Overall, our data suggest that DMF protects from cerebral edema formation during ischemic stroke by targeting interendothelial junctions in an Nrf2-dependent manner, and provide the basis for a completely new approach to treat brain edema.

Differential Cerebrovascular Toxicity of Various Tobacco Products: A Regulatory Perspective.

Blood-Brain Barrier (BBB) is a dynamic anatomical interface that separates brain parenchyma from blood circulation and is principally constituted by the cerebral microcapillary endothelial cells [1,2]. BBB is composed of distinct structural and functional organization through the presence of inter-endothelial tight junction complexes, abundant expression of nutrient and efflux transporters including metabolically active sites. While the TJ complexes tightly seal the paracellular gaps and contribute to high resistance of BBB [3], the presence of specific nutrient transporters and receptor systems selectively regulate the delivery of metabolic substrates, nutrients and macromolecules to the brain. In addition, efflux transporters belonging to the ABC superfamily prevent the brain permeation of blood-borne neurotoxic chemicals including xenobiotics and eliminates the accumulation of toxic metabolites within the brain parenchyma [1]. Taken together, the BBB serves as a physiological, transport and metabolic barrier that critically regulates ion, molecular and cellular flux into the brain, thus maintaining the CNS microenvironment for optimal neuronal function [2]. Importantly, impairment of BBB integrity by various exogenous or endogenous pathological stimuli involving increased load of oxidative/inflammatory stress in the neurovascular unit, is a potential mechanism underlying the pathogenesis of a host of neurologic and degenerative disorders [3,4].

Abstract 166: Matrix Metalloproteinase-9 Mediates Post-hypoxic Vascular Pruning Of Cerebral Blood Vessels By Degrading Laminin And Claudin-5

Objective: Vascular remodeling involves a highly coordinated break-down and build-up of the vascular basal lamina and inter-endothelial tight junction proteins. The goal of this study was to examine the role of matrix metalloproteinase-9 (MMP-9) in remodeling of cerebral blood vessels, both in hypoxia-induced angiogenesis and in the vascular pruning that accompanies the switch from hypoxia back to normoxia. Approach and Results: In a chronic mild hypoxia model of cerebrovascular remodeling, gel zymography revealed that MMP-9 levels were increased, both in the hypoxic angiogenic response and in the post-hypoxic pruning response. Compared to wild-type mice, MMP-9 KO mice showed no alteration in hypoxic-induced angiogenesis, but did show marked delay in post-hypoxic vascular pruning. In wild-type mice, vascular pruning was associated with fragmentation of vascular laminin and the tight junction protein claudin-5, while this process was markedly attenuated in MMP-9 KO mice. In vitro experiments showed that hypoxia stimulated MMP-9 expression in brain endothelial cells (BECs) but not pericytes. While immunofluorescent and flow cytometry analyses showed that hypoxia led to reduced expression of laminin and claudin-5 in wild-type BECs, this decrease was absent in MMP-9 KO BECs. Conclusions: These results show that while MMP-9 is not essential for hypoxic-induced cerebral angiogenesis, it plays an important role in post-hypoxic vascular pruning by degrading laminin and claudin-5. Our data support the concept that MMP-9 inhibition might provide therapeutic benefit in the treatment of ischemic stroke, by preventing post-hypoxic vascular pruning, thereby optimizing vascular density and integrity.