John Anagli

Assessing neuro-systemic & behavioral components in the pathophysiology of blast-related brain injury

Among the U.S. military personnel, blast injury is among the leading causes of brain injury. During the past decade, it has become apparent that even blast injury as a form of mild traumatic brain injury (mTBI) may lead to multiple different adverse outcomes, such as neuropsychiatric symptoms and long-term cognitive disability. Blast injury is characterized by blast overpressure, blast duration, and blast impulse. While the blast injuries of a victim close to the explosion will be severe, majority of victims are usually at a distance leading to milder form described as mild blast TBI (mbTBI). A major feature of mbTBI is its complex manifestation occurring in concert at different organ levels involving systemic, cerebral, neuronal, and neuropsychiatric responses; some of which are shared with other forms of brain trauma such as acute brain injury and other neuropsychiatric disorders such as post-traumatic stress disorder. The pathophysiology of blast injury exposure involves complex cascades of chronic psychological stress, autonomic dysfunction, and neuro/systemic inflammation. These factors render blast injury as an arduous challenge in terms of diagnosis and treatment as well as identification of sensitive and specific biomarkers distinguishing mTBI from other non-TBI pathologies and from neuropsychiatric disorders with similar symptoms. This is due to the “distinct” but shared and partially identified biochemical pathways and neuro-histopathological changes that might be linked to behavioral deficits observed. Taken together, this article aims to provide an overview of the current status of the cellular and pathological mechanisms involved in blast overpressure injury and argues for the urgent need to identify potential biomarkers that can hint at the different mechanisms involved.

Changes in Vascular Permeability and Expression of Different Angiogenic Factors Following Anti-Angiogenic Treatment in Rat Glioma

Background Anti-angiogenic treatments of malignant tumors targeting vascular endothelial growth factor receptors (VEGFR) tyrosine kinase are being used in different early stages of clinical trials. Very recently, VEGFR tyrosine kinase inhibitor (Vetanalib, PTK787) was used in glioma patient in conjunction with chemotherapy and radiotherapy. However, changes in the tumor size, tumor vascular permeability, vascular density, expression of VEGFR2 and other angiogenic factors in response to PTK787 are not well documented. This study was to determine the changes in tumor size, vascular permeability, fractional plasma volume and expression of VEGFR2 in PTK787 treated U-251 glioma rat model by in vivo magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). The findings were validated with histochemical and western blot studies. Methodologies and Principal Findings Seven days after implantation of U251 glioma cells, animals were treated with either PTK787 or vehicle-only for two weeks, and then tumor size, tumor vascular permeability transfer constant (Ktrans), fractional plasma volume (fPV) and expression of VEGFR2 and other relevant angiogenic factors were assessed by in vivo MRI and SPECT (Tc-99-HYNIC-VEGF), and by immunohistochemistry and western blot analysis. Dynamic contrast-enhanced MRI (DCE-MRI) using a high molecular weight contrast agent albumin-(GdDTPA) showed significantly increased Ktrans at the rim of the treated tumors compared to that of the central part of the treated as well as the untreated (vehicle treated) tumors. Size of the tumors was also increased in the treated group. Expression of VEGFR2 detected by Tc-99m-HYNIC-VEGF SPECT also showed significantly increased activity in the treated tumors. In PTK787-treated tumors, histological staining revealed increase in microvessel density in the close proximity to the tumor border. Western blot analysis indicated increased expression of VEGF, SDF-1, HIF-1α, VEGFR2, VEGFR3 and EGFR at the peripheral part of the treated tumors compared to that of central part of the treated tumors. Similar expression patters were not observed in vehicle treated tumors. Conclusion These findings indicate that PTK787 treatment induced over expression of VEGF as well as the Flk-1/VEGFR2 receptor tyrosine kinase, especially at the rim of the tumor, as proven by DCE-MRI, SPECT imaging, immunohistochemistry and western blot.

In vitro MS-based proteomic analysis and absolute quantification of neuronal-glial injury biomarkers in cell culture system.

MS‐based proteomics has been the method of choice for biomarker discovery in the field of traumatic brain injury (TBI). Due to its high sensitivity and specificity, MS is now being explored for biomarker quantitative validation in tissue and biofluids. In this study, we demonstrate the use of MS in both qualitative protein identification and targeted detection of acute TBI biomarkers released from degenerating cultured rat cortical mixed neuronal cells, mimicking intracellular fluid in the central nervous system after TBI. Calpain activation was induced by cell treatment with maitotoxin (MTX), a known calcium channel opener. Separate plates of mixed neuronal‐glial culture were subjected to excitotoxin N‐methyl‐D‐aspartate (NMDA) and apoptotic inducer staurosporine. Acute TBI biomarkers, GFAP and UCH‐L1, were first detected and assessed in the culture media by Western blot. The cell‐conditioned media were then trypsinized and subjected to bottom up proteomic analysis. GFAP was readily detected by data‐dependent scanning but not UCH‐L1. As a proof‐of‐principle study, rat glia‐enriched cell cultures treated with MTX were used to investigate the time‐dependent release of GFAP breakdown product by Western blot and for isotope dilution MS absolute quantitation method development. Absolute quantitation of the GFAP release was conducted using the three cortical mixed neuronal cell cultures treated with different agents. Other differentially expressed proteins identified in the glial‐enriched and cortical mixed neuronal cell culture models were further analyzed by bioinformatic tools. In summary, this study demonstrates the use of MS in both protein identification and targeted quantitation of acute TBI biomarkers and is the preliminary step toward development of TBI biomarker validation by targeted MS.

Integration of Proteomics, Bioinformatics, and Systems Biology in Traumatic Brain Injury Biomarker Discovery

Traumatic brain injury (TBI) is a major medical crisis without any FDA-approved pharmacological therapies that have been demonstrated to improve functional outcomes. It has been argued that discovery of disease-relevant biomarkers might help to guide successful clinical trials for TBI. Major advances in mass spectrometry (MS) have revolutionized the field of proteomic biomarker discovery and facilitated the identification of several candidate markers that are being further evaluated for their efficacy as TBI biomarkers. However, several hurdles have to be overcome even during the discovery phase which is only the first step in the long process of biomarker development. The high-throughput nature of MS-based proteomic experiments generates a massive amount of mass spectral data presenting great challenges in downstream interpretation. Currently, different bioinformatics platforms are available for functional analysis and data mining of MS-generated proteomic data. These tools provide a way to convert data sets to biologically interpretable results and functional outcomes. A strategy that has promise in advancing biomarker development involves the triad of proteomics, bioinformatics, and systems biology. In this review, a brief overview of how bioinformatics and systems biology tools analyze, transform, and interpret complex MS datasets into biologically relevant results is discussed. In addition, challenges and limitations of proteomics, bioinformatics, and systems biology in TBI biomarker discovery are presented. A brief survey of researches that utilized these three overlapping disciplines in TBI biomarker discovery is also presented. Finally, examples of TBI biomarkers and their applications are discussed.

Additivity in Effects of Vitronectin and Monoclonal Antibodies against α-Helix F of Plasminogen Activator Inhibitor-1 on Its Reactions with Target Proteinases

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential therapeutic target in cardiovascular and cancerous diseases. PAI-1 circulates in blood as a complex with vitronectin. A PAI-1 variant (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 PAI-1) with a fluorescent tag at the reactive center loop (RCL) was used to study the effects of vitronectin and monoclonal antibodies (mAbs) directed against α-helix F (Mab-2 and MA-55F4C12) on the reactions of PAI-1 with tissue-type and urokinase-type plasminogen activators. Both mAbs delay the RCL insertion and induce an increase in the stoichiometry of inhibition (SI) to 1.4-9.5. Binding of vitronectin to NBD P9 PAI-1 does not affect SI but results in a 2.0-6.5-fold decrease in the limiting rate constant (klim) of RCL insertion for urokinase-type plasminogen activator at pH 6.2-8.0 and for tissue-type plasminogen activator at pH 6.2. Binding of vitronectin to the complexes of NBD P9 PAI-1 with mAbs results in a decrease in klim and in a 1.5-22-fold increase in SI. Thus, vitronectin and mAbs demonstrated additivity in the effects on the reaction with target proteinases. The same step in the reaction mechanism remains limiting for the rate of RCL insertion in the absence and presence of Vn and mAbs. We hypothesize that vitronectin, bound to α-helix F on the side opposite to the epitopes of the mAbs, potentiates the mAb-induced delay in RCL insertion and the associated substrate behavior by selectively decreasing the rate constant for the inhibitory branch of PAI-1 reaction (ki). These results demonstrate that mAbs represent a valid approach for inactivation of vitronectin-bound PAI-1 in vivo.

Identification of tyrosine nitration in UCH‐L1 and GAPDH

Protein tyrosine nitration is a post‐translational modification commonly used as a marker of cellular oxidative stress associated with numerous pathophysiological conditions. We focused on ubiquitin carboxyl terminal hydrolase‐L1 (UCH‐L1) and glyceraldehyde‐3‐phosphate (GAPDH) which are high‐abundant brain proteins that have been identified to be highly susceptible to oxidative modification. Both UCH‐L1 and GAPDH have been linked to the pathogenesis of Alzheimers and Parkinsons disease, however specific nitration sites have not been elucidated. Identification of specific nitration sites and quantitation of endogenous nitrated proteins are important in correlating this modification to disease pathology. In this study, purified UCH‐L1 and GAPDH were nitrated in vitro with peroxynitrite and the presence of nitrated proteins was confirmed by anti‐3‐nitrotyrosine Western blots. Data‐dependent LC‐MS/MS analysis identified several distinct tyrosine nitration sites in UCH‐L1 (Tyr‐80) and GAPDH (Tyr‐47, Tyr‐92, and Tyr‐312). Subsequent validation with synthetic peptides was conducted for selected nitropeptides. An LC‐MS/MS method was developed for semi‐quantitative determination of the synthetic nitropeptides: KGQEVSPKVY*(UCH‐L1) and mFQY*DSTHGKF (GAPDH). The nitropeptides were detectable in the mid‐attomole range and the peak area response was linear over three orders of magnitude. Targeted analysis of endogenous UCH‐L1 and GAPDH nitration was then conducted in an in vivo second‐hand smoke rat model to evaluate the utility of this approach.

Interactions of Plasminogen Activator Inhibitor-1 with Vitronectin Involve an Extensive Binding Surface and Induce Mutual Conformational Rearrangements†

In order to explore early events during the association of plasminogen activator inhibitor-1 (PAI-1) with its cofactor vitronectin, we have applied a robust strategy that combines protein engineering, fluorescence spectroscopy, and rapid reaction kinetics. Fluorescence stopped-flow experiments designed to monitor the rapid association of PAI-1 with vitronectin indicate a fast, concentration-dependent, biphasic binding of PAI-1 to native vitronectin but only a monophasic association with the somatomedin B (SMB) domain, suggesting that multiple phases of the binding interaction occur only when full-length vitronectin is present. Nonetheless, in all cases, the initial fast interaction is followed by slower fluorescence changes attributed to a conformational change in PAI-1. Complementary experiments using an engineered, fluorescently silent PAI-1 with non-natural amino acids showed that concomitant structural changes occur as well in native vitronectin. Furthermore, we have measured the effect of vitronectin on the rate of insertion of the reactive center loop into beta-sheet A of PAI-1 during reaction with target proteases. With a variety of PAI-1 variants, we observe that both full-length vitronectin and the SMB domain have protease-specific effects on the rate of loop insertion but that the two exhibit clearly different effects. These results support a model for PAI-1 binding to vitronectin in which the interaction surface extends beyond the region of PAI-1 occupied by the SMB domain. In support of this model are recent results that define a PAI-1-binding site on vitronectin that lies outside the somatomedin B domain (Schar, C. R., Blouse, G. E., Minor, K. H., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309) and the complementary site on PAI-1 (Schar, C. R., Jensen, J. K., Christensen, A., Blouse, G. E., Andreasen, P. A., and Peterson, C. B. (2008) J. Biol. Chem. 283, 28487-28496).

Post-Genomics Nanotechnology Is Gaining Momentum: Nanoproteomics and Applications in Life Sciences

The post-genomics era has brought about new Omics biotechnologies, such as proteomics and metabolomics, as well as their novel applications to personal genomics and the quantified self. These advances are now also catalyzing other and newer post-genomics innovations, leading to convergences between Omics and nanotechnology. In this work, we systematically contextualize and exemplify an emerging strand of post-genomics life sciences, namely, nanoproteomics and its applications in health and integrative biological systems. Nanotechnology has been utilized as a complementary component to revolutionize proteomics through different kinds of nanotechnology applications, including nanoporous structures, functionalized nanoparticles, quantum dots, and polymeric nanostructures. Those applications, though still in their infancy, have led to several highly sensitive diagnostics and new methods of drug delivery and targeted therapy for clinical use. The present article differs from previous analyses of nanoproteomics in that it offers an in-depth and comparative evaluation of the attendant biotechnology portfolio and their applications as seen through the lens of post-genomics life sciences and biomedicine. These include: (1) immunosensors for inflammatory, pathogenic, and autoimmune markers for infectious and autoimmune diseases, (2) amplified immunoassays for detection of cancer biomarkers, and (3) methods for targeted therapy and automatically adjusted drug delivery such as in experimental stroke and brain injury studies. As nanoproteomics becomes available both to the clinician at the bedside and the citizens who are increasingly interested in access to novel post-genomics diagnostics through initiatives such as the quantified self, we anticipate further breakthroughs in personalized and targeted medicine.

Different expression of ubiquitin C-terminal hydrolase-L1 and αII-spectrin in ischemic and hemorrhagic stroke: Potential biomarkers in diagnosis.

The two primary categories of stroke, ischemic and hemorrhagic, both have fundamentally different mechanisms and thus different treatment options. These two stroke categories were applied to rat models to identify potential biomarkers that can distinguish between them. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) without reperfusion while hemorrhagic stroke was induced by injecting collagenase IV into the striatum. Brain hemispheres and biofluids were collected at two time points: 3 and 6h after stroke. Known molecules were tested on the rat samples via quantitative immunoblotting (injured brain, CSF) and Banyans proprietary ELISA assays (CSF, serum). The injured brain quantitative analyses revealed that αII-spectrin breakdown products (SBDP150, SBDP145) were strongly increased after 6h ischemia. In CSF, SBDP145 and ubiquitin C-terminal hydrolase-L1 (UCH-L1) levels were elevated after 6h ischemic stroke detected by Western blot and ELISA. In serum UCH-L1 levels were increased after 3 and 6h of ischemia detected by ELISA. However, levels of those proteins in hemorrhagic stroke remain normal. In summary, in both the brain and the biofluids, SBDPs and UCH-L1 were elevated after ischemic but not hemorrhagic stroke. These molecules behaved differently in the two stroke models and thus may be capable of being differentiated.

A neuroproteomic and systems biology analysis of rat brain post intracerebral hemorrhagic stroke

Intracerebral hemorrhage (ICH) is a devastating form of stroke leading to a high rate of death and disability worldwide. Although it has been hypothesized that much of the IHC insult occurs in the subacute period mediated via a series of complex pathophysiological cascades, the molecular mechanisms involved in ICH have not been systematically characterized. Among the best approaches to understand the underlying mechanisms of injury and recovery, protein dynamics assessment via proteomics/systems biology platforms represent one of the cardinal techniques optimized for mechanisms investigation and biomarker identification. A proteomics approach may provide a biomarker focused framework from which to identify candidate biomarkers of pathophysiological processes involved in brain injury after stroke. In this work, a neuroproteomic approach (LC-MS/MS) was applied to investigate altered expression of proteins that are induced in brain tissue 3 h after injury in a rat model of ICH. Data from sham and focal ischemic models were also obtained and used for comparison. Based on the differentially expressed protein profile, systems biology analysis was conducted to identify associated cellular processes and related interaction maps. After LC-MS/MS analysis of the 3 h brain lysates, 86 proteins were differentially expressed between hemorrhagic and sham tissues. Furthermore, 38 proteins were differentially expressed between ischemic and sham tissues. On the level of global pathway analysis, hemorrhagic stroke proteins were shown to be involved in autophagy, ischemia, necrosis, apoptosis, calpain activation, and cytokine secretion. Moreover, ischemic stroke proteins were related to cell death, ischemia, inflammation, oxidative stress, caspase activation and apoptotic injury. In conclusion, the proteomic responses identified in this study provide key information about target proteins involved in specific pathological pathways.