Kimberly E. Hawkins

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

BackgroundMatrix 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.ResultsA 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.ConclusionsWe 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.

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.

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 90u2009minutes and were killed at 4, 14, 24, and 48u2009hours 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 16u2009hours. 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

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 that reduces neuroinflammation in stroke. Since LXA4 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.

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.

Genetic Mechanisms of Memory Disorders (Excluding Alzheimer's Disease)

In this chapter we consider human disorders of memory and their possible underlying genetic and molecular mechanisms. The genetic and molecular basis of Neurofibromatosis type I and other RASopathies, Angelman syndrome, Fragile X syndrome, Williams–Beuren syndrome, Down syndrome, Pitt-Hopkins syndrome, and Rett syndrome will be discussed. An overview of current research into suitable models of disease and treatment will provide insight into the value of basic scientific research in understanding human memory disorders and intellectual disabilities.

Chapter 23 – Epigenetics of Memory Processes

Epigenetic mechanisms that help regulate gene activity in the central nervous system have historically been thought to be involved exclusively in developmental processes or in disease states. However, recent work argues that these mechanisms, particularly posttranslational modifications of histones and covalent modification of DNA, remain labile through out the life span and are altered by experiences. Indeed, exciting new data indicate that epigenetic regulation of genes is necessary for experience-induced changes in adult brain function and behavior. In this chapter, we focus on this theme and review the data implicating epigenetic mechanisms in neural plasticity and memory formation. As this is still considered a relatively new transcriptional regulatory mechanism in the context of memory formation, we also discuss some of the caveats and limitations of the data.

Abstract CT085: iCare 1: A prospective clinical trial to predict treatment response based on mutanome-informed computational biology in patients with AML and MDS

Hypomethylating agents (HMAs) (azacitidine (aza), decitabine (dec)) and lenalidomide (len) are approved agents and used to treat patients with myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). Despite their widespread use, HMAs fail in the majority of these patients, and len fails in 75% of non-del(5q) MDS. Currently, no method exists to predict disease response, thus the management of MDS and AML patients is challenging. Methods: Patients with AML or MDS were recruited to a clinical trial (NCT02435550) designed to assess predictive values by comparing computer predictions of drug response to actual clinical response. Genomic profiling was conducted by cytogenetics, whole exome sequencing, and array CGH. Genomic results were inputted into a computational software (Cellworks), which generates disease-specific protein network maps using PubMed and other resources. Digital drug simulations were conducted by quantitatively measuring drug effect on a cell growth score (proliferation + viability + apoptosis). Each patient-specific protein network was screened for the extent by which aza, dec or len reduced disease growth in a dose-respondent manner. Treatment was physician’s choice of SOC. Clinical outcomes were prospectively recorded. IWG criteria were used to define response. Western blot assays were performed to validate the predicted protein network perturbations. Fisher’s exact test was used to compare prediction values of the genomics-informed computer method versus empiric drug administration. Results: 88 patients have had all molecular tests and computational modeling performed. Lab validation of computer-predicted, activated protein networks in 19 samples from 13 different patients showed correct prediction of 5 activated networks (Akt2, Akt3, PIK3CA, p38, Erk1/2) in 17 samples, with 89% accuracy. At the time of this report, 26/88 patients were eligible for efficacy evaluation. 8/26 patients showed clinical response to SOC therapy, 18/26 did not. 24/26 outcome predictions were correctly matched to their clinical outcomes, and 2/20 were incorrectly matched, resulting in 92% prediction accuracy, 80% PPV, 100% NPV, 100% sensitivity, and 89% specificity. The accuracy of the genomics-informed computer method was significantly greater than empiric drug administration (p=1.664e-05). New genomic signature rules were discovered to correlate with clinical response after aza, dec or len. Summary: A computational method that models multiple genomic abnormalities simultaneously showed high predictive value of protein network aberrations and clinical outcomes after SOC treatments. The network method uncovered molecular reasons for drug failure and highlighted resistance pathways that could be targeted to recover chemosensitivity. This technology could also be used to establish eligibility criteria for precision enrollment in drug development trials Citation Format: Leylah Drusbosky, Kimberly E. Hawkins, Shireen Vali, Taher Abbasi, Ansu Kumar, Neeraj Kumar Singh, Kabya Basu, Chandan Kumar, Amjad Husain, Caitlin Tucker, Randy A. Brown, Maxim Norkin, John Hiemenz, Jack Hsu, John Wingard, Christopher R. Cogle. iCare 1: A prospective clinical trial to predict treatment response based on mutanome-informed computational biology in patients with AML and MDS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT085. doi:10.1158/1538-7445.AM2017-CT085