John B. Redell

The consequences of disrupting cardiac inwardly rectifying K+ current (IK1) as revealed by the targeted deletion of the murine Kir2.1 and Kir2.2 genes

1 Ventricular myocytes demonstrate a steeply inwardly rectifying K+ current termed IK1. We investigated the molecular basis for murine IK1 by removing the genes encoding Kir2.1 and Kir2.2. The physiological consequences of the loss of these genes were studied in newborn animals because mice lacking Kir2.1 have a cleft palate and die shortly after birth. 2 Kir2.1 ‐/‐ ventricular myocytes lack detectable IK1 in whole‐cell recordings in 4 mM external K+. In 60 mM external K+ a small, slower, residual current is observed. Thus Kir2.1 is the major determinant of IK1. Sustained outward K+ currents and Ba2+ currents through L‐ and T‐type channels were not significantly altered by the mutation. A 50 % reduction in IK1 was observed in Kir2.2‐/‐ mice, raising the possibility that Kir2.2 can also contribute to the native IK1. 3 Kir2.1 ‐/‐ myocytes showed significantly broader action potentials and more frequent spontaneous action potentials than wild‐type myocytes. 4 In electrocardiograms of Kir2.1‐/‐ neonates, neither ectopic beats nor re‐entry arrhythmias were observed. Thus the increased automaticity and prolonged action potential of the mutant ventricular myocytes were not sufficiently severe to disrupt the sinus pacing of the heart. The Kir2.1‐/‐ mice, however, had consistently slower heart rates and this phenotype is likely to arise indirectly from the influence of Kir2.1 outside the heart. 5 Thus Kir2.1 is the major component of murine IK1 and the Kir2.1‐/‐ mouse provides a model in which the functional consequences of removing IK1 can be studied at both cellular and organismal levels.

Enhancing Expression of Nrf2-Driven Genes Protects the Blood–Brain Barrier after Brain Injury

The integrity of the blood–brain barrier (BBB) is critical for normal brain function, and its compromise contributes to the pathophysiology of a number of CNS diseases and injuries. Using a rodent model of brain injury, the present study examines the pathophysiology of BBB disruption. Western blot and immunohistochemical analyses indicate that brain injury causes a loss of capillary endothelial cells and tight junction proteins, two critical components of the BBB. Activation of the transcription factor NF-E2-related factor-2 (Nrf2) by sulforaphane, a naturally occurring compound present in high levels in cruciferous vegetables, significantly increased the expression of endogenous cytoprotective genes in brain tissue and microvessels as indicated by real-time PCR analysis. Postinjury administration of sulforaphane reduced the loss of endothelial cell markers and tight junction proteins and preserved BBB function. These protective effects were dependent on the activity of Nrf2. Injured rats pretreated with decoy oligonucleotides containing the binding site of Nrf2, and mice lacking the nrf2 gene, did not benefit from sulforaphane administration. These findings indicate a potential therapeutic usefulness for Nrf2-activating molecules to improve the function of the neurovascular unit after injury.

Human Traumatic Brain Injury Alters Plasma microRNA Levels

Circulating microRNAs (miRNAs) present in the serum/plasma are characteristically altered in many pathological conditions, and have been employed as diagnostic markers for specific diseases. We examined if plasma miRNA levels are altered in patients with traumatic brain injury (TBI) relative to matched healthy volunteers, and explored their potential for use as diagnostic TBI biomarkers. The plasma miRNA profiles from severe TBI patients (Glasgow Coma Scale [GCS] score ≤8) and age-, gender-, and race-matched healthy volunteers were compared by microarray analysis. Of the 108 miRNAs identified in healthy volunteer plasma, 52 were altered after severe TBI, including 33 with decreased and 19 with increased relative abundance. An additional 8 miRNAs were detected only in the TBI plasma. We used quantitative RT-PCR to determine if plasma miRNAs could identify TBI patients within the first 24 h post-injury. Receiver operating characteristic curve analysis indicated that miR-16, miR-92a, and miR-765 were good markers of severe TBI (0.89, 0.82, and 0.86 AUC values, respectively). Multiple logistic regression analysis revealed that combining these miRNAs markedly increased diagnostic accuracy (100% specificity and 100% sensitivity), compared to either healthy volunteers or orthopedic injury patients. In mild TBI patients (GCS score > 12), miR-765 levels were unchanged, while the plasma levels of miR-92a and miR-16 were significantly increased within the first 24 h of injury compared to healthy volunteers, and had AUC values of 0.78 and 0.82, respectively. Our results demonstrate that circulating miRNA levels are altered after TBI, providing a rich new source of potential molecular biomarkers. Plasma-derived miRNA biomarkers, used in combination with established clinical practices such as imaging, neurocognitive, and motor examinations, have the potential to improve TBI patient classification and possibly management.

Traumatic Brain Injury Alters Expression of Hippocampal MicroRNAs: Potential Regulators of Multiple Pathophysiological Processes

Multiple cellular, molecular, and biochemical changes contribute to outcome after traumatic brain injury (TBI). MicroRNAs (miRNAs) are known to influence many important cellular processes, including proliferation, apoptosis, neurogenesis, angiogenesis, and morphogenesis, all processes that are involved in TBI pathophysiology. However, it has not yet been determined whether miRNA expression is altered after TBI. In the present study, we used a microarray platform to examine changes in the hippocampal expression levels of 444 verified rodent miRNAs at 3 and 24 hr after controlled cortical impact injury. Our analysis found 50 miRNAs exhibited decreased expression levels and 35 miRNAs exhibited increased expression levels in the hippocampus after injury. We extended the microarray findings using quantitative polymerase chain reaction analysis for a subset of the miRNAs with altered expression levels (miR‐107, ‐130a, ‐223, ‐292‐5p, ‐433‐3p, ‐451, ‐541, and ‐711). Bioinformatic analysis of the predicted targets for this panel of miRNAs revealed an overrepresentation of proteins involved in several biological processes and functions known to be initiated after injury, including signal transduction, transcriptional regulation, proliferation, and differentiation. Our results indicate that multiple protein targets and biological processes involved in TBI pathophysiology may be regulated by miRNAs.

Biomarkers for the Diagnosis and Prognosis of Mild Traumatic Brain Injury/Concussion

Mild traumatic brain injury (mTBI) results from a transfer of mechanical energy into the brain from traumatic events such as rapid acceleration/deceleration, a direct impact to the head, or an explosive blast. Transfer of energy into the brain can cause structural, physiological, and/or functional changes in the brain that may yield neurological, cognitive, and behavioral symptoms that can be long-lasting. Because mTBI can cause these symptoms in the absence of positive neuroimaging findings, its diagnosis can be subjective and often is based on self-reported neurological symptoms. Further, proper diagnosis can be influenced by the motivation to conceal or embellish signs and/or an inability of the patient to notice subtle dysfunctions or alterations of consciousness. Therefore, appropriate diagnosis of mTBI would benefit from objective indicators of injury. Concussion and mTBI are often used interchangeably, with concussion being primarily used in sport medicine, whereas mTBI is used in reference to traumatic injury. This review provides a critical assessment of the status of current biomarkers for the diagnosis of human mTBI. We review the status of biomarkers that have been tested in TBI patients with injuries classified as mild, and introduce a new concept for the discovery of biomarkers (termed symptophenotypes) to predict common and unique symptoms of concussion. Finally, we discuss the need for biomarker/biomarker signatures that can detect mTBI in the context of polytrauma, and to assess the consequences of repeated injury on the development of secondary injury syndrome, prolongation of post-concussion symptoms, and chronic traumatic encephalopathy.

Identification of serum biomarkers in brain-injured adults: potential for predicting elevated intracranial pressure.

Brain injury biomarkers may have clinical utility in stratifying injury severity level, predicting adverse secondary events or outcomes, and monitoring the effectiveness of therapeutic interventions. As a biomarker source, serum offers several advantages over cerebrospinal fluid (CSF), including ease of accessibility and reduced risk to the patient. We screened pooled serum samples obtained from 11 severely injured traumatic brain injury (TBI) patients (Glasgow Coma Scale [GCS] <or= 8) and 11 age-, sex- and race-matched volunteers. Two time points-41.5 +/- 4.9 h and 66.3 +/- 6.6 h post-injury-were chosen for the initial screening analysis. Samples were immunodepleted for 12 highly abundant serum proteins, and then labeled with mass-balanced isobaric tags (iTRAQ), and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Identification and quantification of 2455 iTRAQ-labeled peptides that mapped to 160 proteins revealed 31 candidate biomarkers whose serum abundance was altered after injury. Changes in three candidate biomarkers (serum amyloid A, [SAA], c-reactive protein [CRP], retinol binding protein 4 [RBP4]) were verified using independent TBI and healthy volunteer serum samples. Receiver operating characteristic (ROC) curve analysis of CRP and SAA indicated they were robust indicators of injury even at very acute time points. Analysis of serum RBP4 levels at 24-36 h post-injury indicates it may predict subsequent increases in intracranial pressure (ICP) with a sensitivity of 86% and specificity of 88% at 11.6 mug/mL [n = 7, ICP < 20 mm Hg; n = 8, ICP > 25 mm Hg). Our results support the use of serum as a source for discovery of TBI biomarkers, and indicate that serum biomarkers may have utility for predicting secondary pathologies (e.g., elevated ICP) associated with TBI.

Biomarkers in the clinical diagnosis and management of traumatic brain injury.

Traumatic brain injury (TBI) is the leading cause of death and disability among young adults. Numerous safety improvements in the workplace, the addition of airbags to vehicles, and the enforcement of speed limits have all helped to reduce the incidence and severity of head trauma. While improvements in emergency response times and acute care have increased TBI survivability, this has heightened the necessity for developing reliable methods to identify patients at risk of developing secondary pathologies. At present, the primary clinical indicators for the presence of brain injury are the Glasgow Coma Scale (GCS), pupil reactivity, and head computed tomography (CT). While these indices have proven useful for stratifying the magnitude and extent of brain damage, they have limited utility for predicting adverse secondary events or detecting subtle damage. Biomarkers, reflecting a biological response to injury or disease, have proven useful for the diagnosis of many pathological conditions including cancer, heart failure, infection, and genetic disorders. For TBI, several proteins synthesized in astroglial cells or neurons have been proposed as potential biomarkers. These proteins include the BB isozyme of creatine kinase (CK-BB, predominant in brain), glial fibrilary acidic protein (GFAP), myelin basic protein (MBP), neuron-specific enolase (NSE), and S100B.The presence of these biomarkers in the cerebrospinal fluid and serum of patients with moderate-to-severe TBI, and their correlation with outcome, suggest that they may have utility as surrogate markers in clinical trials. In addition, many of these markers have been found to be sensitive indicators of injury, and therefore may have the potential to diagnose persons with mild TBI. In addition to biomarkers that correlate with long-term outcome, a few studies have identified prognostic biomarkers for secondary injury that may be useful in individualizing patient management.

Human mesenchymal stem cells inhibit vascular permeability by modulating vascular endothelial cadherin/β-catenin signaling.

The barrier formed by endothelial cells (ECs) plays an important role in tissue homeostasis by restricting passage of circulating molecules and inflammatory cells. Disruption of the endothelial barrier in pathologic conditions often leads to uncontrolled inflammation and tissue damage. An important component of this barrier is adherens junctions, which restrict paracellular permeability. The transmembrane protein vascular endothelial (VE)-cadherin and its cytoplasmic binding partner β-catenin are major components of functional adherens junctions. We show that mesenchymal stem cells (MSCs) significantly reduce endothelial permeability in cocultured human umbilical vascular endothelial cells (HUVECs) and following exposure to vascular endothelial growth factor, a potent barrier permeability-enhancing agent. When grown in cocultures with HUVECs, MSCs increased VE-cadherin levels and enhanced recruitment of both VE-cadherin and β-catenin to the plasma membrane. Enhanced membrane localization of β-catenin was associated with a decrease in β-catenin-driven gene transcription. Disruption of the VE-cadherin/β-catenin interaction by overexpressing a truncated VE-cadherin lacking the β-catenin interacting domain blocked the permeability-stabilizing effect of MSCs. Interestingly, a conditioned medium from HUVEC-MSC cocultures, but not from HUVEC or MSC cells cultured alone, significantly reduced endothelial permeability. In addition, intravenous administration of MSCs to brain-injured rodents reduced injury-induced enhanced blood-brain barrier permeability. Similar to the effect on in vitro cultures, this stabilizing effect on blood-brain barrier function was associated with increased expression of VE-cadherin. Taken together, these results identify a putative mechanism by which MSCs can modulate vascular EC permeability. Further, our results suggest that the mediator(s) of these vascular protective effects is a secreted factor(s) released as a result of direct MSC-EC interaction.

Mesenchymal Stem Cells Regulate Blood-Brain Barrier Integrity Through TIMP3 Release After Traumatic Brain Injury

The matrix metalloproteinase inhibitor TIMP3 mediates the beneficial effects of mesenchymal stem cells on the blood-brain barrier of the injured mouse brain. Mesenchymal Stem Cells Spill Their Secrets Traumatic brain injury (TBI) is the leading cause of death and disability in children and young adults worldwide and is considered a “silent epidemic” in the United States in both civilian and military populations. Pathological cerebral edema and blood-brain barrier (BBB) permeability are the leading causes of death acutely after TBI with very few therapeutic options. It has been established in animal models that intravenously administered adult bone marrow–derived mesenchymal stem cells (MSCs) are able to ameliorate BBB permeability in mice after TBI. In new work, Menge et al. identify the mechanism responsible for this beneficial effect and identify the mediator as a soluble factor produced by MSCs called TIMP3. In a mouse model of TBI, Menge et al. show that down-regulation of TIMP3 expression in intravenously administered human MSCs abrogates their protective effects on the BBB and endothelial cell stability after TBI. Furthermore, the authors demonstrate that administering intravenous recombinant human TIMP3 alone to mice after TBI can fully recapitulate the protective effects of MSCs on vascular stability and BBB integrity, indicating that TIMP3 may be a key factor regulating integrity of the BBB. Although much more work needs to be done, TIMP3 could be a useful cell-free therapeutic for treating the breakdown of BBB integrity and cerebral edema that occurs after TBI. Mesenchymal stem cells (MSCs) may be useful for treating a variety of disease states associated with vascular instability including traumatic brain injury (TBI). A soluble factor, tissue inhibitor of matrix metalloproteinase-3 (TIMP3), produced by MSCs is shown to recapitulate the beneficial effects of MSCs on endothelial function and to ameliorate the effects of a compromised blood-brain barrier (BBB) due to TBI. Intravenous administration of recombinant TIMP3 inhibited BBB permeability caused by TBI, whereas attenuation of TIMP3 expression in intravenously administered MSCs blocked the beneficial effects of the MSCs on BBB permeability and stability. MSCs increased circulating concentrations of soluble TIMP3, which blocked vascular endothelial growth factor-A–induced breakdown of endothelial cell adherens junctions in vitro and in vivo. These findings elucidate a potential molecular mechanism for the beneficial effects of MSCs on the BBB after TBI and demonstrate a role for TIMP3 in the regulation of BBB integrity.

Analysis of Functional Pathways Altered after Mild Traumatic Brain Injury

Concussive injury (or mild traumatic brain injury; mTBI) can exhibit features of focal or diffuse injury patterns. We compared and contrasted the cellular and molecular responses after mild controlled cortical impact (mCCI; a focal injury) or fluid percussion injury (FPI; a diffuse injury) in rats. The rationale for this comparative analysis was to investigate the brains response to mild diffuse versus mild focal injury to identify common molecular changes triggered by these injury modalities and to determine the functional pathways altered after injury that may provide novel targets for therapeutic intervention. Microarrays containing probes against 21,792 unique messenger RNAs (mRNAs) were used to investigate the changes in cortical mRNA expression levels at 3 and 24 h postinjury. Of the 354 mRNAs with significantly altered expression levels after mCCI, over 89% (316 mRNAs) were also contained within the mild FPI (mFPI) data set. However, mFPI initiated a more widespread molecular response, with over 2300 mRNAs differentially expressed. Bioinformatic analysis of annotated gene ontology molecular function and biological pathway terms showed a significant overrepresentation of genes belonging to inflammation, stress, and signaling categories in both data sets. We therefore examined changes in the protein levels of a panel of 23 cytokines and chemokines in cortical extracts using a Luminex-based bead immunoassay and detected significant increases in macrophage inflammatory protein (MIP)-1α (CCL3), GRO-KC (CXCL1), interleukin (IL)-1α, IL-1β, and IL-6. Immunohistochemical localization of MIP-1α and IL-1β showed marked increases at 3 h postinjury in the cortical vasculature and microglia, respectively, that were largely resolved by 24 h postinjury. Our findings demonstrate that both focal and diffuse mTBI trigger many shared pathobiological processes (e.g., inflammatory responses) that could be targeted for mechanism-based therapeutic interventions.