Neuroinflammatory mechanisms of blood-brain barrier damage in ischemic stroke.

As part of the neurovascular unit, the blood-brain barrier (BBB) is a unique, dynamic regulatory boundary that limits and regulates the exchange of molecules, ions, and cells between the blood and the central nervous system. Disruption of the BBB plays an important role in the development of neurological dysfunction in ischemic stroke. Blood-borne substances and cells have restricted access to the brain due to the presence of tight junctions between the endothelial cells of the BBB. Following stroke, there is loss of BBB tight junction integrity, leading to increased paracellular permeability, which results in vasogenic edema, hemorrhagic transformation, and increased mortality. Thus, understanding principal mediators and molecular mechanisms involved in BBB disruption is critical for the development of novel therapeutics to treat ischemic stroke. This review discusses the current knowledge of how neuroinflammation contributes to BBB damage in ischemic stroke. Specifically, we provide an updated overview of the role of cytokines, chemokines, oxidative and nitrosative stress, adhesion molecules, matrix metalloproteinases, and vascular endothelial growth factor as well as the role of different cell types in the regulation of BBB permeability in ischemic stroke.

Neurovascular protection by post-ischemic intravenous injections of the lipoxin A4 receptor agonist, BML-111, in a rat model of ischemic stroke.

Resolution of inflammation is an emerging new strategy to reduce damage following ischemic stroke. Lipoxin A4 (LXA4 ) is an anti-inflammatory, pro-resolution lipid mediator with high affinity binding to ALX, the lipoxin A4 receptor. Since LXA4 is rapidly inactivated, potent analogs have been created, including the ALX agonist BML-111. We hypothesized that post-ischemic intravenous administration of BML-111 would provide protection to the neurovascular unit and reduce neuroinflammation in a rat stroke model. Animals were subjected to 90 min of middle cerebral artery occlusion (MCAO) and BML-111 was injected 100 min and 24 h after stroke onset and animals euthanized at 48 h. Post-ischemic treatment with BML-111 significantly reduced infarct size, decreased vasogenic edema, protected against blood-brain barrier disruption, and reduced hemorrhagic transformation. Matrix metalloproteinase-9 and matrix metalloproteinase-3 were significantly reduced following BML-111 treatment. Administration of BML-111 dramatically decreased microglial activation, as seen with CD68, and neutrophil infiltration and recruitment, as assessed by levels of myeloperoxidase and intracellular adhesion molecule-1. The tight junction protein zona occludens-1 was protected from degradation following treatment with BML-111. These results indicate that post-ischemic activation of ALX has pro-resolution effects that limit the inflammatory damage in the cerebral cortex and helps maintain blood-brain barrier integrity after ischemic stroke.

A genomics-informed computational biology platform prospectively predicts treatment responses in AML and MDS patients.

Patients with myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) are generally older and have more comorbidities. Therefore, identifying personalized treatment options for each patient early and accurately is essential. To address this, we developed a computational biology modeling (CBM) and digital drug simulation platform that relies on somatic gene mutations and gene CNVs found in malignant cells of individual patients. Drug treatment simulations based on unique patient-specific disease networks were used to generate treatment predictions. To evaluate the accuracy of the genomics-informed computational platform, we conducted a pilot prospective clinical study (NCT02435550) enrolling confirmed MDS and AML patients. Blinded to the empirically prescribed treatment regimen for each patient, genomic data from 50 evaluable patients were analyzed by CBM to predict patient-specific treatment responses. CBM accurately predicted treatment responses in 55 of 61 (90%) simulations, with 33 of 61 true positives, 22 of 61 true negatives, 3 of 61 false positives, and 3 of 61 false negatives, resulting in a sensitivity of 94%, a specificity of 88%, and an accuracy of 90%. Laboratory validation further confirmed the accuracy of CBM-predicted activated protein networks in 17 of 19 (89%) samples from 11 patients. Somatic mutations in the TET2, IDH1/2, ASXL1, and EZH2 genes were discovered to be highly informative of MDS response to hypomethylating agents. In sum, analyses of patient cancer genomics using the CBM platform can be used to predict precision treatment responses in MDS and AML patients.

Using social media to assess care coordination goals and plans for leukemia patients and survivors.

Care coordination has been shown to have a positive effect on the management of chronic disease. Specific to the management of leukemia, coordination may occur between primary care physician, medical and radiation oncologists, surgeons, cardiologists, and genetics specialists. Experiencing gaps in communication and care coordination, many health consumers seek instrumental support in their social circles, including online forums and networks. The goal of this theory-guided study was to provide an in-depth assessment of how individuals use online forums to deliberate about their goals and plans for leukemia care coordination. Guided by the planning theory of communication, the data were collected from the American Cancer Society Cancer Survivors Network and included 125 original posts and 1,248 responses. Thematic analysis and axial coding were applied to analyze the data. Goal-related themes included overcoming the diffusion of care coordination and achieving health management cohesion. Planning themes included social health management, communication self-efficacy, and role deliberation. Online patient forums provide an interactive platform for patients and caregivers to engage in active conversations, which in turn can serve as identifiers of care coordination needs. Communication with those who share similar experiences allows cancer patients and survivors to accumulate functional health literacy, gain communication self-efficacy, and articulate a care coordination role acceptable to them.

Spatiotemporal Changes in P-glycoprotein Levels in Brain and Peripheral Tissues Following Ischemic Stroke in Rats.

P-glycoprotein (P-gp) is known to transport a diverse array of xenobiotics, including therapeutic drugs. A member of the ATP-binding cassette (ABC) transporter family, P-gp is a protein encoded by the gene Mdr1 in humans and Abcb1 in rodents (represented by 2 isoforms Abcb1a and Abcb1b). Lining the luminal and abluminal membrane of brain capillary endothelial cells, P-gp is a promiscuous efflux pump extruding a variety of exogenous toxins and drugs. In this study, we measured dynamic changes in Abcb1a and Abcb1b transcripts and P-gp protein in the brain, liver, and kidney after experimental stroke. P-glycoprotein has been shown to increase in brain endothelial cells following hypoxia in vitro or after exposure to proinflammatory cytokines. Using a rat model of ischemic stroke, we hypothesized that P-gp expression will be increased in the brain, liver, and kidney in response to neuroinflammation following ischemic stroke. Adult Sprague Dawley rats underwent middle cerebral artery occlusion (MCAO) for 90 minutes and were killed at 4, 14, 24, and 48 hours postreperfusion onset to determine the time course of P-gp expression. To mimic ischemia occurring at the blood-brain barrier, rat brain endothelial (RBE4) cells were subjected to hypoxia and low glucose (HLG) for 16 hours. Immunoblotting analyses showed P-gp increases in brain and liver following 90-minute MCAO, as well as in cultured RBE4 cells after 16-hour HLG treatment, but fluctuated in the kidney depending on the time point. The relative roles of each isoform in the protein expression were analyzed with quantitative reverse transcriptase polymerase chain reaction. Ischemic stroke leads to significant increases in P-gp levels not only in the brain but also in the liver. The increase in P-gp could dramatically reduce the bioavailability and efficacy of neuroprotective drugs. Therefore, P-gp represents a big hurdle to drug delivery to the ischemic brain.

Targeting resolution of neuroinflammation after ischemic stroke with a lipoxin A4 analog: Protective mechanisms and long-term effects on neurological recovery.

BACKGROUND: Resolution of inflammation is an emerging new strategy to reduce damage following ischemic stroke. Lipoxin A4 (LXA 4) is an anti-inflammatory, pro-resolution lipid mediator that reduces neuroinflammation in stroke. Since LXA 4 is rapidly inactivated, potent analogs have been synthesized, including BML-111. We hypothesized that post-ischemic, intravenous treatment with BML-111 for 1 week would provide neuroprotection and reduce neurobehavioral deficits at 4 weeks after ischemic stroke in rats. Additionally, we investigated the potential protective mechanisms of BML-111 on the post-stroke molecular and cellular profile. METHODS: A total of 133 male Sprague-Dawley rats were subjected to 90 min of transient middle cerebral artery occlusion (MCAO) and BML-111 administration was started at the time of reperfusion. Two methods of week-long BML-111 intravenous administration were tested: continuous infusion via ALZET ® osmotic pumps (1.25 and 3.75 µg µl-1 hr-1), or freshly prepared daily single injections (0.3, 1, and 3 mg/kg). We report for the first time on the stability of BML-111 and characterized an optimal dose and a dosing schedule for the administration of BML-111. RESULTS: One week of BML-111 intravenous injections did not reduce infarct size or improve behavioral deficits 4 weeks after ischemic stroke. However, post-ischemic treatment with BML-111 did elicit early protective effects as demonstrated by a significant reduction in infarct volume and improved sensorimotor function at 1 week after stroke. This protection was associated with reduced pro-inflammatory cytokine and chemokine levels, decreased M1 CD40+ macrophages, and increased alternatively activated, anti-inflammatory M2 microglia/macrophage cell populations in the post-ischemic brain. CONCLUSION: These data suggest that targeting the endogenous LXA 4 pathway could be a promising therapeutic strategy for the treatment of ischemic stroke. More work is necessary to determine whether a different dosing regimen or more stable LXA 4 analogs could confer long-term protection.

Computational drug treatment simulations on projections of dysregulated protein networks derived from the myelodysplastic mutanome match clinical response in patients.

Although the majority of MDS patients fail to achieve clinical improvement to approved therapies, some patients benefit from treatment. Predicting patient response prior to therapy would improve treatment effectiveness, avoid treatment-related adverse events and reduce healthcare costs. Three separate cohorts of MDS patients were used to simulate drug response to lenalidomide alone, hypomethylating agent (HMA) alone, or HMA plus lenalidomide. Utilizing a computational biology program, genomic abnormalities in each patient were used to create an intracellular pathway map that was then used to screen for drug response. In the lenalidomide treated cohort, computer modeling correctly matched clinical responses in 37/46 patients (80%). In the second cohort, 15 HMA patients were modeled and correctly matched to responses in 12 (80%). In the third cohort, computer modeling correctly matched responses in 10/10 patients (100%). This computational biology network approach identified GGH overexpression as a potential resistance factor to HMA treatment and paradoxical activation of beta-catenin (through Csnk1a1 inhibition) as a resistance factor to lenalidomide treatment. We demonstrate that a computational technology is able to map the complexity of the MDS mutanome to simulate and predict drug response. This tool can improve understanding of MDS biology and mechanisms of drug sensitivity and resistance.

The molecular neurobiology of the sleep-deprived, fuzzy brain.

Sleep deprivation is well established to cause diminution of cognitive function, including disruption of both minute-to-minute working memory and decrements in the stabilization of long-term memories. Moreover, "replay" during sleep of episodes and sequences of events that were experienced during wakefulness has been implicated in consolidation of long-term memories. However, the molecular mechanisms underlying the role of sleep in memory function are just starting to be defined. In this issue of Science Signaling, Tudor et al identify one molecular component underlying the effects of sleep on memory function: dynamic experience-dependent regulation of protein synthesis in the hippocampus.

Adropin reduces paracellular permeability of rat brain endothelial cells exposed to ischemia-like conditions.

Adropin is a peptide encoded by the energy homeostasis associated gene (Enho) and plays a critical role in the regulation of lipid metabolism, insulin sensitivity, and endothelial function. Little is known of the effects of adropin in the brain and whether this peptide modulates ischemia-induced blood-brain barrier (BBB) injury. Here, we used an in vitro BBB model of rat brain microvascular endothelial cells (RBE4) and hypothesized that adropin would reduce endothelial permeability during ischemic conditions. To mimic ischemic conditions in vitro, RBE4 cell monolayers were subjected to 16h hypoxia/low glucose (HLG). This resulted in a significant increase in paracellular permeability to FITC-labeled dextran (40kDa), a dramatic upregulation of vascular endothelial growth factor (VEGF), and the loss of junction proteins occludin and VE-cadherin. Notably, HLG also significantly decreased Enho expression and adropin levels. Treatment of RBE4 cells with synthetic adropin (1, 10 and 100ng/ml) concentration-dependently reduced endothelial permeability after HLG, but this was not mediated through protection to junction proteins or through reduced levels of VEGF. We found that HLG dramatically increased myosin light chain 2 (MLC2) phosphorylation in RBE4 cells, which was significantly reduced by adropin treatment. We also found that HLG significantly increased Rho-associated kinase (ROCK) activity, a critical upstream effector of MLC2 phosphorylation, and that adropin treatment attenuated that effect. These data indicate that treatment with adropin reduces endothelial cell permeability after HLG insult by inhibition of the ROCK-MLC2 signaling pathway. These promising findings suggest that adropin protects against endothelial barrier dysfunction during ischemic conditions.

Detrimental role of the EP1 prostanoid receptor in blood-brain barrier damage following experimental ischemic stroke.

Cyclooxygenase-2 (COX-2) is activated in response to ischemia and significantly contributes to the neuroinflammatory process. Accumulation of COX-2-derived prostaglandin E2 (PGE2) parallels the substantial increase in stroke-mediated blood-brain barrier (BBB) breakdown. Disruption of the BBB is a serious consequence of ischemic stroke, and is mainly mediated by matrix metalloproteinases (MMPs). This study aimed to investigate the role of PGE2 EP1 receptor in neurovascular injury in stroke. We hypothesized that pharmacological blockade or genetic deletion of EP1 protects against BBB damage and hemorrhagic transformation by decreasing the levels and activity of MMP-3 and MMP-9. We found that post-ischemic treatment with the EP1 antagonist, SC-51089, or EP1 genetic deletion results in a significant reduction in BBB disruption and reduced hemorrhagic transformation in an experimental model of transient focal cerebral ischemia. These neurovascular protective effects of EP1 inactivation are associated with a significant reduction in MMP-9/-3, less peripheral neutrophil infiltration, and a preservation of tight junction proteins (ZO-1 and occludin) composing the BBB. Our study identifies the EP1 signaling pathway as an important link between neuroinflammation and MMP-mediated BBB breakdown in ischemic stroke. Targeting the EP1 receptor could represent a novel approach to diminish the devastating consequences of stroke-induced neurovascular damage.

Fluorometric immunocapture assay for the specific measurement of matrix metalloproteinase-9 activity in biological samples: application to brain and plasma from rats with ischemic stroke.

BACKGROUND: Matrix metalloproteinases are important factors in the molecular mechanisms leading to neuronal injury in many neurological disorders. Matrix metalloproteinase (MMP)-9 is up-regulated after cerebral ischemia and neuroinflammation and is actively involved in blood-brain barrier disruption. Current methods of measuring MMP-9 activity, such as gelatin-substrate zymography, are unspecific and arduous. Here we developed an immunocapture assay with high efficiency, specificity, and sensitivity for quantifying endogenously active as well as total MMP-9 activity. RESULTS: A fluorescence resonance energy transfer (FRET) peptide-based immunocapture assay was developed that enables the accurate assessment of total and active forms of MMP-9 in complex biological samples. The FRET assay demonstrated correct and efficient binding of MMP-9 to a mouse monoclonal MMP-9 antibody and high specificity of the immunocapture antibody for MMP-9. Total and active levels of MMP-9 were measured in rat brain homogenates, plasma, human HT-1080 conditioned media, and RBE4 endothelial cell lysates. The FRET immunocapture assay yielded highly similar results for total MMP-9 activity when compared to gelatin-substrate zymography. CONCLUSIONS: We suggest that the new FRET peptide-based immunocapture assay is a viable replacement of zymography for sensitive and high throughput quantification of MMP-9 activity in biological samples.