Center for Blood-Brain Barrier Research
The members of the Center for Blood-Brain Barrier Research include 12 PIs from three Departments (Biomedical Sciences, Immunotherapeutics & Biotechnology, and Pharmaceutical Sciences) and their teams (currently 17 Graduate students and five postdocs).
The Center's mission is to discover and develop novel therapeutics that will advance drug therapy in diseases affecting the central nervous system. The Center will foster research to characterize new drug targets based on understanding of both receptor and transport mechanisms and of metabolic pathways.
By encouraging collaboration with colleagues across School of Pharmacy departments, the Center will raise the pharmacy school's research profile, enhance the competitiveness of Center members for extramural funding and increase the attractiveness of the TTUHSC School of Pharmacy to prospective faculty members.
Members of the Center and Their Research Interests
Please click on a name to link to each faculty member's individual web page.
Dr. Abbruscato’s group investigates the role of the glucose carrier GLUT1 and of the Sodium Dependent Glucose Transporter (SGLT) at the blood-brain barrier (BBB). In particular, they focus on the regulation of SGLT under pathophysiological conditions such as ischemia/reperfusion injury (stroke). Selective pharmacological modulation of SGLT could have clinical implications: (i) inhibition of SGLT at the BBB could be beneficial in stroke by limiting excessive glucose delivery to brain and brain edema (because SGLT transfers 210 molecules of water for each Na+ ion and glucose molecule); (ii) modulating SGLT activity in neurons could be protective by reducing oxidative stress and cellular edema; Further studies on the expression and regulation of SGLT in vivo with temporary focal ischemia combined with reperfusion would lead to a better understanding as to how it can be utilized as a therapeutic target in stroke and other CNS disorders. These studies are funded by the National Institutes of Neurological Disorders and Stroke in collaboration with Drs. Bickel and Smith.
Based on substantial work in his lab and by others, he believes that the Na+, K+, 2Cl- Cotransporter (NKCC) plays a critical role in cellular edema associated with stroke. NKCC protein expression and function has been proven to be modulated by PKC in ischemia in our lab and could provide a promising drug target. One exciting avenue to pursue is the �cross-talk� between NKCC and opioid receptors. Classically, opioid receptor activation stimulates K+ channels and suppresses Ca2+ current into the cytoplasm of neurons. Opioid receptor activation during stoke has been shown to modulate other key ion channels by the activation of protein kinase C (PKC). Publications from Drs. Abbruscato and Karamyan labs have also shown that the selective δ agonist, DPDPE, μ agonist, DAMGO and δ/μ receptor agonist, biphalin, significantly decrease water gain induced in hippocampus slices subjected to oxygen-glucose deprivation. Biphalin has also been shown to reduce infarction and edema ratios, penumbral injury, and neuronal NKCC activation via PKC (Yang et al., JPET 2011; Yang et al., Brain Research 2011). Current experiments in collaboration with Dr. Karamyan are characterizing the regional expression of opioid receptor types in the ischemic brain during ischemia-reperfusion injury. Structure activity experiments are being conducted in collaboration with Dr. Victor Hruby (University of Arizona. Department of Chemistry) characterizing novel opioid agonists that are designed to target the predominant opioid receptors expressed during stroke injury. Multi-functional opioid receptor agonists are being designed with a variety of pharmacophores positioned differently by a variety of linking strategies.
The spectrum of methods in Dr. Abbruscato’s laboratory includes the following in vitro and in vivo techniques:
Models of the BBB (bovine primary, mouse cell line); hypoxia chamber for reduced oxygen/glucose experiments; trans-endothelial cell resistance measurement; mouse primary neuronal and astrocytic cell culture; hippocampal brain slicing and oxygen glucose deprivation experiments; Western blot analysis. Available in vivo techniques include permanent and reversible mouse MCAO; infarct and edema ratio; locomotor testing for neurologic outcome post ischemia; Blood-CNS pharmacokinetics; in situ brain perfusion, genetic and experimental models of diabetes mellitus; mouse or rat brain endothelial cell isolation.
The main focus of Dr. Bickel’s lab is the identification of novel, BBB-specific targets for drug delivery. Receptor systems like the transferrin receptor or insulin receptor are highly expressed at the BBB and have been successfully used in preclinical studies for brain targeting, but these receptors are obviously not brain-specific. A vascular targeting strategy based on receptors only expressed at the BBB has thus far been elusive. This is an attractive goal, because it may reduce the risk of unwanted effects in peripheral organs, and potentially allow regional targeting to brain tissue affected by disease (e.g., brain tumors or neuroinflammatory conditions associated with ischemia/reperfusion injury). We are collaborating with Dr. Jon Weidanz (Immunotherapy Center, Texas Tech School of Pharmacy, Abilene) on the characterization of peptide-MHC complexes on brain endothelial cells as unique targets for specific T-cell receptor mimic antibodies (TCRm). These studies received initial funding by a NIH R21 grant. A current collaborative project (with Dr. Jon Weidanz and Dr. Paul Lockman, TTUHSC School of Pharmacy, Amarillo) explores the potential therapeutic effect of TCRm on brain metstasis of breast cancer. It is supported by an IDEA Award of the Breast Cancer Research Program (DoD).
Another project in Dr. Bickel’s group addresses the question, whether protection of the BBB by targeted delivery of anti-inflammatory drugs to brain microvascular endothelial cells can be beneficial in stroke therapy. Our studies in models of transient and permanent brain ischemia in mice showed significant therapeutic effect of anti-inflammatory oligonucleotide drugs when delivered via BBB transferrin receptors. Currently our goal is to characterize at the cellular level, where the pharmacological effect seen in the whole animal models actually occur (e.g., activity on endothelial cells, neurons, and/or glial cells?). This research is supported by a grant from the American Heart Association. In addition to the middle cerebral artery occlusion (MCAO) stroke model, we are using brain microdialysis to monitor pharmacological effects in vivo under stroke conditions.
The following methods are routinely used in Dr. Bickel’s laboratory:
In vitro techniques:
Brain endothelial cell culture (cell lines from human and murine origin); production and purification of monoclonal antibodies from hybridoma supernatant; transport studies with endothelial cell monolayers (Transwell); cell adhesion assays ; flowcytometry ; radiotracer labeling; radioligand binding assays; ELISA; Northern blotting; Real time PCR; gel shift assays; HPLC and FPLC for analytical and semipreparative purpose; protein conjugations; supravital staining of brain slices (TTC); immunohistochemistry; wide field fluorescence microscopy and confocal microscopy.
Ex vivo methods:
Autoradiography of brain cryostat sections following administration of radioactive tracers in vivo; capillary depletion technique to characterize brain uptake; brain capillary and endothelial cell isolation.
In vivo techniques:
Middle Cerebral Artery Occlusion (MCAO) by intraluminal suture for induction of permanent or transient brain ischemia in mice; brain microdialysis; Laser Doppler Flowmetry; carotid artery catheterization in mice (and rats) for arterial blood sampling in pharmacokinetic studies; carotid artery catheterization for vascular brain perfusion; sensorimotor testing in mice (Corner test).
Dr. Cucullo’s group research focus investigates three areas of research revolving around the physiology and pathophysiology of the blood-brain barrier: 1) Pathophysiology of tobacco smoke toxicity at the neurovascular unit. The goal of this research project is to investigate both in vivo (rodent models) and in vitro differential effects of tobacco/cigarette smoke exposure from “reduced-exposure” versus regular cigarette brands, and to identify potential prodromic factors for the pathogenesis and progression of cerebrovascular impairment and brain disorders. The study encompasses cross-interaction between systemic inflammation and neuronal activity and involves In vitro and In vivo assessment of peripheral blood immune cells interaction via inflammatory mediators with the blood-brain barrier endothelium as well as pathogenesis and progression of secondary brain injuries. The study makes use of immunohistochemistry and electroencephalographic monitoring techniques (brain microsurgery and stereotactic electrode placement) as well as brain histology, and characterization by FACS analysis and cell sorting of sub-populations of circulating white blood cells.
2) In vitro modeling of the human cerebrovascular system. The goal of this research study is to optimize current hollow fiber technology with the purpose of developing a dynamic (flow-based) artificial models of the human brain vascular network to enable/facilitate the study of the effect of hemodynamic (e.g., stroke, transient ischemia) and pro-inflammatory challenges on BBB integrity and function as well as provide an ideal artificial testing and optimization platform for rationale CNS drug design and delivery methods. The study encompasses the characterization of the pharmacokinetic and pharmacodynamic properties of the BBB including mechanisms of pharmacoresistance; BBB induction and maintenance; evaluation of BBB function in response to endogenous and exogenous stimuli (e.g., inflammation, stroke, hypoperfusion, ischemia, brain trauma, etc).
3) Role of Altered Glycemia in the Pathogenesis of CNS Disorders through alterations of BBB function and integrity. A hallmark of the chronic complications associated with diabetes mellitus (particularly type 2) is compromised microvascular integrity. The complexity of the processes that cause CNS disease in diabetes and the difficulty of detecting vascular impairments in the large and heterogeneous brain microvascular bed make the study and characterization of this pathology extremely difficult. The main goal of this study is to assess the impact and unfold the mechanism/s by which hyper- and hypoglycemia can impair BBB integrity and function.
General Methods in use in Dr.Cucullo’s lab include the following:
Models of the BBB (bovine primary, human primary, Human cell lines, Rodent (rat) cell line); hypoxia chamber for reduced oxygen/glucose experiments; real time trans-endothelial cell resistance measurement; Static (Transwell) and flow-based (based on capillary-like hollow fiber technology) BBB multi-culture models; In vitro flow cessation-reperfusion w/wo circulating immune cells; Western blot analysis, ELISA, Immunocytochemistry; online real time NO, H2O2, and glucose measurements, etc.
Our group is studying nature and function of the brain renin-angiotensin system, and its regulation at the level of receptors and proteolytic enzymes in health and disease. In the last several years our main research efforts were focused on pharmacological and biochemical characterization of a novel, non-AT1, non-AT2 angiotensin binding site discovered in our laboratory. Most recently, we identified the novel angiotensin binding site as membrane-bound variant of metalloendopeptidase neurolysin (EC 188.8.131.52). Our current studies are focused on understanding the (patho)physiological role of neurolysin in neurogenic hypertension and ischemic stroke.
Additionally, in collaboration with Dr. Tom Abbruscato we study the role of opioid receptors for neuroprotection in stroke. This project involves (patho)physiological and pharmacological studies to understand alterations of opioid receptors in pathophysiology of stroke, and development of new opioid ligands (in collaboration with medicinal chemists) to target these receptors.
The following techniques/methods are regularly used in our laboratory:
primary brain cultures (neuronal, astroglial and capillary endothelial), cell-viability assays, radioligand receptor binding assays (whole cell and membrane), functional assays for GPCRs, in vitro autoradiography, immunoblotting, immunocytochemistry, fluorescence microscopy, protein modification and purification, proteolytic studies, enzymatic assays, supravital staining of brain slices (TTC), middle cerebral artery occlusion (MCAO) by intraluminal suture for induction of permanent or transient brain ischemia in mice, Laser Doppler Flowmetry; sensorimotor testing in mice; blood pressure monitoring in mice and rats using radiotelemetry, dosing and sampling methods in rats and mice.
Dr. Lockman’s research is focused evaluating various drug therapies to treat and prevent brain metastases of breast cancer. Brain metastases pose a life threatening problem for women with advanced metastatic breast cancer. Of women who have been diagnosed with disseminated breast cancer, ~10-16% will develop symptomatic brain metastases and at least 20-30% will have micrometastatic lesions present at autopsy. For patients with metastatic Her-2 amplified or “triple negative” breast tumors, ~30-45% of patients will develop brain metastases, often as the first site of relapse when they are responding to systemic chemotherapy or experiencing stable disease. Once lesions are established in the central nervous system, only one in five women survive one year. We have recently shown that the blood-tumor barrier and blood-brain barrier, although significantly compromised, still markedly restrict drug delivery and inhibit chemotherapeutic induced cytotoxicty in ~90% of CNS metastases. Our work has been funded by the Department of Defense, a number of drug companies and the NIH.
Major techniques in the laboratory include:
A model of brain metastases of breast cancer, orthotopic brain cancer models, in vivo blood-brain pharmacokinetics, in situ brain perfusions, autoradiography, quantitative fluorescent microscopy, immunofluorescence, nanoparticle and liposomal formulations, primary and immortalized cell cultures of brain and cancer cells, HPLC analysis of drug molecules.
Dr. Mehvar’s research is focused on the pharmacological approaches to reduce ischemia-reperfusion (IR) injury in the liver. Hepatic ischemia-reperfusion injury is a serious, yet so far unavoidable, complication of many clinical situations such as liver transplantation, major liver resection surgery (Pringle maneuver), and hemorrhagic and septic shock. Although all of the in vivo conditions resulting in the interruption of blood supply to the liver produce warm IR injury, liver transplantation is unique in that, in addition to a variable degree of warm IR injury during organ retrieval and/or transplantation, it is also subjected to cold (storage) IR injury during organ preservation. Initially, we focused on the pharmacokinetic-based delivery approaches to target anti-inflammatory drugs to the liver for prevention of IR injury. Our current emphasis, however, is on the role of cytochrome P450 in generation of reactive oxygen species (ROS) and pharmacological approaches to inhibit P450 generation of ROS.
Part of this project is the live-cell imaging of hypoxia/reoxygenation injury in hepatocytes by confocal microscopy, which recently stimulated collaboration with Dr. Bickel’s lab.
Major techniques used in our laboratory are:
In vivo warm hepatic IR model in rats; ex vivo warm hepatic IR model in rats; ex vivo cold hepatic IR model in rats; rat liver transplantation model; macromolecular-based drug delivery to the liver; primary cultures of rat hepatocytes; P450 content and activity assays; HPLC analysis of small molecules and macromolecules in biological samples; pharmacokinetic dosing and sampling methods in rodents; and pharmacokinetic and drug metabolism data analysis.
The members of this laboratory are using various in vivo and in vitro approaches to study brain metabolism.
1. Epilepsy. In collaboration with Dr Karin Borges, Univ Queensland, Australia, we showed an anaplerotic diet rich in triheptanoin could alter seizure parameters in two mouse models of epilepsy. Current research is focused on identifying the molecular mechanisms of this protective response. Pharmacological manipulations of metabolic pathways will allow us to identify pathways that are affected by triheptanoin and determine if they are involved in the antiepileptic response. Further, we showed that voluntary exercise in mice alters seizure development in the pilocarpine/status epilepticus model. Ongoing experiments are testing if exercise and triheptanoin share a common mechanism of action.
2. Lipocalin-2 (LCN2). This iron-binding protein is induced as part of the innate immune response and is induced early in epileptogenesis. In an in vitro model, a necrotic cell extract can induce LCN2 in the C6 astroglioma cell line. Experiments are underway to identify the mechanism of induction. As inflammation contributes to the development of epilepsy, a long-term goal of this project is to identify mechanisms by which LCN2 contributes to the development of or protection from epilepsy.
3. Choline transporter. Choline is an essential nutrient important for growth and maintenance of cells. The protein responsible for uptake into cells has not been convincingly demonstrated. We are using chemical and molecular biological approaches to identify this protein. Present research involves development of a photoaffinity probe to identify the transporter. Once identified, our long-term goals are to identify the role of this protein in tissue injury and repair.
A wide range of experiment approaches and techniques are in use in the laboratory. We are currently using in vivo models for epilepsy. There is extensive experience in primary neural cell culture along with use of culture cell lines. Molecular expertise includes all routine biochemical and molecular techniques such as chromatography, cloning and expression of proteins, quantitative PCR, immunoblotting and histochemistry, microscopy, and RNA silencing.
The main focus of Dr. Trippier’s research is drug discovery and medicinal chemistry. The group uses the principles of rational and in silico drug design to generate compounds with desirable activity. The techniques of synthetic medicinal chemistry are used to probe the structure-activity relationship of the molecules and enhance the observed biological activity. Of specific interest is the design of highly specific and potent kinase inhibitors, a class of enzyme that represents excellent targets for therapeutic intervention of a range of neurodegenerative diseases such as Alzheimer’s and the neuromuscular disease Spinal Muscular Atrophy. The group is also interested in designing small molecule probes to elucidate the complex signaling pathways involved in axon regeneration.
Major techniques used in our laboratory include: Organic synthesis; compound purification; chemical biology; HPLC; nuclear magnetic resonance structure elucidation; mass spectrometry; molecular modeling; enzyme-based assays; cell-based assays.
With the collaboration of Dr. Ulrich Bickel (TTUHSC) and Dr. George Gokel (University of Missouri, St. Louis), I recently submitted an RO1 proposal to synthesize and test a series of compounds that are predicted to increase eNOS activity. Our preliminary studies showed that triacsin C, the lead compound in the series, inhibits long chain fatty acyl CoA synthetase, inhibits eNOS palmitoylation, increases NO production in both cultured endothelial cells and intact aorta, and enhances methacholine induced relaxation of vascular smooth muscle. It is anticipated that these compounds may have clinical utility in treatment of hypertension. There is preliminary evidence that triacsin C and like compounds may have neuroprotective effects in an experimental model of stroke. In both a temporary (1 hour of occlusion followed by 24 hours reperfusion) and permanent (24 hours occlusion) stroke model, triacsin C significantly reduced the cerebral infarct volume. Other data indicates that triacsin C may not only increase eNOS activity, it may also inhibit iNOS expression. Hypertension is the single most important risk factor for stroke. It is estimated that about 80% of stroke victims are hypertensive. Our observations with triacsin C suggest that it is possible to develop an antihypertensive drug that would also have the benefit of reducing neurologic damage in the event of a stroke. The Ischemia/Reperfusion Injury Center would provide an important mechanism for feedback and discussion of this research.
Major Biochemical Techniques used in my laboratory include: Assay of catalytic activities of long chain fatty acyl CoA synthetase and nitric oxide synthase, radioimmunoassay of prostaglandins, Griess reaction, and extraction and analysis of total tissue/cellular lipid. We also do cell culture (human and rat coronary endothelial cells), endothelial shear stress measurement, western blotting, immunoprecipitation, and siRNA. In addition, I have extensive experience with and am fully equipped to do the Langendorf perfused heart preparation.
Information on this page is current as of February 2013 >/em>