Preclinical Injury Models Research Articles

2016 Scientific Sessions Sol Sherry Distinguished Lecturer in Thrombosis: Thrombotic Stroke: Neuroprotective Therapy by Recombinant-Activated Protein C.

APC (activated protein C), derived from the plasma protease zymogen, is antithrombotic and anti-inflammatory. In preclinical injury models, recombinant APC provides neuroprotection for multiple injuries, including ischemic stroke. APC acts directly on brain endothelial cells and neurons by initiating cell signaling that requires multiple receptors. Two or more major APC receptors mediate APC's neuroprotective cell signaling. When bound to endothelial cell protein C receptor, APC can cleave protease-activated receptor 1, causing biased cytoprotective signaling that reduces ischemia-induced injury. Pharmacological APC alleviates bleeding induced by tissue-type plasminogen activator in murine ischemic stroke studies. Remarkably, APC's signaling promotes neurogenesis. The signaling-selective recombinant variant of APC, 3K3A-APC, was engineered to lack most of the APC's anticoagulant activity but retain APC's cell signaling actions. Recombinant 3K3A-APC is in ongoing National Institutes of Health (NIH)-funded clinical trials for ischemic stroke.

High Dose Meclizine Prevents Renal Ischemia–Reperfusion Injury in Healthy Male Mice

High Dose Meclizine Prevents Renal Ischemia–Reperfusion Injury in Healthy Male Mice

Perioperative elafin for ischaemia-reperfusion injury during coronary artery bypass graft surgery: a randomised-controlled trial

BackgroundElafin is a potent endogenous neutrophil elastase inhibitor that protects against myocardial inflammation and injury in preclinical models of ischaemic-reperfusion injury. We investigated whether elafin could inhibit myocardial ischaemia-reperfusion injury...

Extending injury- and disease-resistant CNS phenotypes by repetitive epigenetic conditioning.

Significant reductions in the extent of acute injury in the CNS can be achieved by exposure to different preconditioning stimuli, but the duration of the induced protective phenotype is typically short-lasting, and thus is deemed as limiting its clinical applicability. Extending the period over which such adaptive epigenetic changes persist – in effect, expanding conditioning’s “therapeutic window” – would significantly broaden the potential applications of such a treatment approach in patients. The frequency of the conditioning stimulus may hold the key. While transient (1–3 days) protection against CNS ischemic injury is well established preclinically following a single preconditioning stimulus, repetitively presenting preconditioning stimuli extends the duration of ischemic tolerance by many weeks. Moreover, repetitive intermittent postconditioning enhances post-ischemic recovery metrics and improves long-term survival. Intermittent conditioning is also efficacious for preventing or delaying injury in preclinical models of chronic neurodegenerative disease, and for promoting long-lasting functional improvements in a number of other pathologies as well. Although the detailed mechanisms underlying these protracted kinds of neuroplasticity remain largely unstudied, accumulating empirical evidence supports the contention that all of these adaptive phenotypes are epigenetically mediated. Going forward, additional preclinical demonstrations of the ability to induce sustained beneficial phenotypes that reduce the burden of acute and chronic neurodegeneration, and experimental interrogations of the regulatory constructs responsible for these epigenetic responses, will accelerate the identification of not only efficacious but also practical, adaptive epigenetics-based treatments for individuals with neurological disease.

PERI-OPERATIVE ELAFIN FOR ISCHAEMIA REPERFUSION INJURY DURING CORONARY ARTERY BYPASS GRAFT/ CARDIAC SURGERY

Elafin is a neutrophil elastase inhibitor that protects against myocardial injury in pre-clinical models of ischemic-reperfusion injury. We investigated if elafin could inhibit myocardial injury induced during coronary artery bypass graft (CABG) surgery. In a randomized double blind placebo

Pathologic mechanical stress and endotoxin exposure increases lung endothelial microparticle shedding.

Acute lung injury (ALI) results from infectious challenges and from pathologic lung distention produced by excessive tidal volume delivered during mechanical ventilation (ventilator-induced lung injury [VILI]) and is characterized by extensive alveolar and vascular dysfunction. Identification of novel ALI therapies is hampered by the lack of effective ALI/VILI biomarkers. We explored endothelial cell (EC)-derived microparticles (EMPs) (0.1-1 μm) as potentially important markers and potential mediators of lung vascular injury in preclinical models of ALI and VILI. We characterized EMPs (annexin V and CD31 immunoreactivity) produced from human lung ECs exposed to physiologic or pathologic mechanical stress (5 or 18% cyclic stretch [CS]) or to endotoxin (LPS). EC exposure to 18% CS or to LPS resulted in increased EMP shedding compared with static cells (∼ 4-fold and ∼ 2.5-fold increases, respectively). Proteomic analysis revealed unique 18% CS-derived (n = 10) and LPS-derived EMP proteins (n = 43). VILI-challenged mice (40 ml/kg, 4 h) exhibited increased plasma and bronchoalveolar lavage CD62E (E-selectin)-positive MPs compared with control mice. Finally, mice receiving intratracheal instillation of 18% CS-derived EMPs displayed significant lung inflammation and injury. These findings indicate that ALI/VILI-producing stimuli induce significant shedding of distinct EMP populations that may serve as potential ALI biomarkers and contribute to the severity of lung injury.

Complement activation in progressive renal disease.

Chronic kidney disease (CKD) is common and the cause of significant morbidity and mortality. The replacement of functioning nephrons by fibrosis is characteristic of progressive disease. The pathways that lead to fibrosis are not fully understood, although chronic non-resolving inflammation in the kidney is likely to drive the fibrotic response that occurs. In patients with progressive CKD there is histological evidence of inflammation in the interstitium and strategies that reduce inflammation reduce renal injury in pre-clinical models of CKD. The complement system is an integral part of the innate immune system but also augments adaptive immune responses. Complement activation is known to occur in many diverse renal diseases, including glomerulonephritis, thrombotic microangiopathies and transplant rejection. In this review we discuss current evidence that complement activation contributes to progression of CKD, how complement could cause renal inflammation and whether complement inhibition would slow progression of renal disease.

A new method for generating high purity motoneurons from mouse embryonic stem cells.

A common problem with using embryonic stem (ES) cells as a source for analysis of gene expression, drug toxicity, or functional characterization studies is the heterogeneity that results from many differentiation protocols. The ability to generate large numbers of high purity differentiated cells from pluripotent stem cells could greatly enhance their utility for in vitro characterization studies and transplantation in pre-clinical injury models. Population heterogeneity is particularly troublesome for post-mitotic neurons, including motoneurons, because they do not proliferate and are quickly diluted in culture by proliferative phenotypes, such as glia. Studies of motoneuron biology and disease, in particular amyotrophic lateral sclerosis, can benefit from high purity motoneuron cultures. In this study, we engineered a transgenic-ES cell line where highly conserved enhancer elements for the motoneuron transcription factor Hb9 were used to drive puromycin N-acetyltransferase expression in ES cell-derived motoneurons. Antibiotic selection with puromycin was then used to obtain high purity motoneuron cultures following differentiation of mouse ES cells. Purity was maintained during maturation allowing the production of consistent, uniform populations of cholinergic ES cell-derived motoneurons. Appropriate functional properties of purified motoneurons were verified by acetylcholinesterase activity and electrophysiology. Antibiotic selection, therefore, can provide an inexpensive alternative to current methods for isolating ES cell-derived motoneurons at high purity that does not require specialized laboratory equipment and provides a unique platform for studies in motoneuron development and degeneration.

Elucidating the severity of preclinical traumatic brain injury models: a role for functional assessment?

Concussion remains a symptom-based diagnosis clinically, yet preclinical studies investigating traumatic brain injury, of which concussion is believed to represent a "mild" form, emphasize histological end points with functional assessments often minimized or ignored all together. Recently, clinical studies have identified the importance of cognitive and neuropsychiatric symptoms, in addition to somatic concerns, following concussion. How these findings may translate to preclinical studies is unclear at present. To address the contrasting end points used clinically compared with those in preclinical studies and the potential role of functional assessments in a commonly used model of diffuse axonal injury (DAI). Animals were subjected to DAI by the use of the impact-acceleration model. Functional and behavioral assessments were conducted during 1 week following DAI before the completion of the histological assessment at 1 week post-DAI. We show, despite the suggestion that this model represents concussive injury, no functional impairments as determined by using the common measures of motor, sensorimotor, cognitive, and neuropsychiatric function following injury over the course of 1 week. The lack of functional deficits is in sharp contrast to neuropathological findings indicating neural degeneration, astrocyte reactivity, and microglial activation. Future studies are needed to identify functional assessments, neurophysiologic techniques, and imaging assessments more apt to distinguish differences following so-called "mild" traumatic brain injury in preclinical models and determine whether these models are truly studying concussive or subconcussive injury. These studies are needed not only to understand the mechanism of injury and production of subsequent deficits, but also to rigorously evaluate potential therapeutic agents.

Human multipotent adult progenitor cells transcriptionally regulate fucosyltransferase VII

Background aimsTargeted recruitment of leukocytes to sites of inflammation is a crucial event in normal host defense against pathogens, and attachment to and rolling on activated endothelial cells is a prerequisite first step for eventual leukocyte extravasation into sites of inflammation. These key events are mediated by interactions between glycosylated ligands expressed on leukocytes and selectins expressed on activated endothelium. Cell surface expression of selectin ligands on leukocytes is regulated by the rate-limiting enzyme fucosyltransferase VII (Fut7), and in its absence extravasation of leukocytes is severely inhibited. Multipotent adult progenitor cells (MAPCs) are an adherent cell population isolated from adult bone marrow. Intravenous administration of MAPCs provided functional improvement in multiple pre-clinical models of injury or disease, but the mechanisms by which these outcomes were achieved remain poorly understood. MethodsIn vitro cell analysis studies including fluorescence-activated cell sorting, messenger RNA analysis, T-cell proliferation assays and endothelial cell binding assays were performed. ResultsThe in vitro cell analysis studies characterized the ability of MAPCs to secrete factors that transcriptionally attenuate expression of Fut7 in T cells, blocking the terminal fucosylation event in the biosynthesis of selectin ligands and reducing T-cell binding to endothelial cells. ConclusionsThis study presents the first example of a distinct regulatory mechanism involving transcriptional down-regulation of Fut7 by MAPCs that could modulate the trafficking behavior of T cells in vivo.

Human multipotent adult progenitor cells modulate leukocyte extravasation by transcriptional regulation of fucosyltransferase VII (P5142)

Abstract The targeted recruitment of leukocytes to sites of inflammation is a crucial event in normal host defense against pathogens, and attachment to and rolling on activated endothelial cells is a prerequisite first step required for eventual leukocyte extravasation into sites of inflammation. These first key events are mediated by interactions between sialylated ligands expressed on leukocytes and selectins expressed on activated endothelium. Cell surface expression of sialylated ligands on leukocytes is regulated by the rate-limiting enzyme fucosyltransferase VII (Fut7), and in its absence extravasation of leukocytes is severely inhibited. Intravenous administration of multipotent adult progenitor cells (MAPC), an adherent cell isolated from adult bone marrow, provides functional improvement in multiple preclinical models of injury or disease, but the mechanism(s) by which these outcomes are achieved remain poorly understood. In this manuscript, in vitro data are presented that characterize the ability of MAPC to secrete factor(s) that transcriptionally attenuate expression of Fut7 in T cells, thereby blocking sialylation of leukocyte cell surface-expressed selectin ligands and reducing T cell binding to endothelial cells. This represents the first example of a distinct regulatory mechanism involving transcriptional down-regulation of Fut7 by MAPC that could modulate the trafficking behavior of T cells in vivo.

Preoperative diet impacts the adipose tissue response to surgical trauma

Short-term changes in preoperative nutrition can have profound effects on surgery-related outcomes such as ischemia/reperfusion injury in preclinical models. Dietary interventions that lend protection against stress in animal models (eg, fasting, dietary restriction [DR]) impact adipose tissue quality/quantity. Adipose tissue holds high surgical relevance because of its anatomic location and large tissue volume, and it is ubiquitously traumatized during surgery. Yet the response of adipose tissue to trauma under clinically relevant circumstances including dietary status remains poorly defined. We hypothesized that preoperative diet alters the adipose tissue response to surgical trauma. A novel mouse model of adipose tissue surgical trauma was employed. Dietary conditions (diet-induced obesity [DIO], preoperative DR) were modulated before application of surgical adipose tissue trauma in the context of clinically common scenarios (different ages, simulated bacterial wound contamination). Local/distant adipose tissue phenotypic responses were measured as represented by gene expression of inflammatory, tissue remodeling/growth, and metabolic markers. Surgical trauma had a profound effect on adipose tissue phenotype at the site of trauma. Milder but significant distal effects on non-traumatized adipose tissue were also observed. DIO exacerbated the inflammatory aspects of this response, and preoperative DR tended to reverse these changes. Age and lipopolysaccharide (LPS)-simulated bacterial contamination also impacted the adipose tissue response to trauma, with young adult animals and LPS treatment exacerbating the proinflammatory response. Surgical trauma dramatically impacts both local and distal adipose tissue biology. Short-term preoperative DR may offer a strategy to attenuate this response.

Decreased Risk of Radiation Pneumonitis With Incidental Concurrent Use of Angiotensin-Converting Enzyme Inhibitors and Thoracic Radiation Therapy

Angiotensin-converting enzyme (ACE) inhibitors have been shown to mitigate radiation-induced lung injury in preclinical models. The aim of this study was to evaluate whether ACE inhibitors decrease the risk of radiation pneumonitis in lung cancer patients receiving thoracic irradiation. Patients with Stage I through III small-cell and non-small-cell lung cancer treated definitively with radiation from 2004-2009 at the Clement J. Zablocki Veterans Affairs Medical Center were retrospectively reviewed. Acute pulmonary toxicity was quantified within 6 months of completion of treatment according to the Common Terminology Criteria for Adverse Events version 4. The use of ACE inhibitors, nonsteroidal anti-inflammatory drugs, inhaled glucocorticosteroids, statins, and angiotensin receptor blockers; dose-volume histogram parameters; and patient factors were assessed for association with Grade 2 or higher pneumonitis. A total of 162 patients met the criteria for inclusion. The majority of patients had Stage III disease (64%) and received concurrent chemotherapy (61%). Sixty-two patients were identified as ACE inhibitor users (38%). All patients had acceptable radiation plans based on dose-volume histogram constraints (V20 [volume of lung receiving at least 20 Gy] ≤37% and mean lung dose ≤20 Gy) with the exception of 2 patients who did not meet both criteria. Grade 2 or higher pulmonary toxicity occurred in 12 patients (7.4%). The rate of Grade 2 or higher pneumonitis was lower in ACE inhibitor users vs. nonusers (2% vs. 11%, p = 0.032). Rates of Grade 2 or higher pneumonitis were significantly increased in patients aged greater than 70 years (16% vs. 2%, p = 0.005) or in whom V5 (volume of lung receiving at least 5 Gy) was 50% or greater (13% vs. 4%, p = 0.04). V10 (volume of lung receiving at least 10 Gy), V20, V30 (volume of lung receiving at least 30 Gy), and mean lung dose were not independently associated with Grade 2 or higher pneumonitis. ACE inhibitors may decrease the incidence of radiation pneumonitis in patients receiving thoracic radiation for lung cancer. These findings are consistent with preclinical evidence and should be prospectively evaluated.

Angiotensin Converting Enzyme Inhibitor Mitigation of Radiation Pneumonitis

ACE inhibitors have been shown to mitigate radiation induced lung injury in preclinical models. The exact mechanisms of action have not been completely elucidated. Proposed mechanisms include alteration of the parenchymal and endothelial response to radiation injury through suppression of the renin-angiotensin system, reductions in TGF-B, and/or alterations in blood flow. The aim of the study was to evaluate the effect of ACE inhibitors on the risk of radiation pneumonitis in lung cancer patients receiving thoracic irradiation.

The potential effects of anti-diabetic medications on myocardial ischemia–reperfusion injury

Heart disease and stroke account for 65% of the deaths in people with diabetes mellitus (DM). DM and hyperglycemia cause systemic inflammation, endothelial dysfunction, a hypercoagulable state with impaired fibrinolysis and increased platelet degranulation, and reduced coronary collateral blood flow. DM also interferes with myocardial protection afforded by preconditioning and postconditioning. Newer anti-diabetic agents should not only reduce serum glucose and HbA1c levels, but also improve cardiovascular outcomes. The older sulfonylurea agent, glyburide, abolishes the benefits of ischemic and pharmacologic preconditioning, but newer sulfonylurea agents, such as glimepiride, may not interfere with preconditioning. GLP-1 analogs and sitagliptin, an oral dipeptidyl peptidase IV inhibitor, limit myocardial infarct size in animal models by increasing intracellular cAMP levels and activating protein kinase A, whereas metformin protects the heart by activating AMP-activated protein kinase. Both thiazolidinediones (rosiglitazone and pioglitazone) limit infarct size in animal models. The protective effect of pioglitazone is dependent on downstream activation of cytosolic phospholipase A(2) and cyclooxygenase-2 with subsequent increased production of 15-epi-lipoxin A(4), prostacyclin and 15-d-PGJ(2). We conclude that agents used to treat DM have additional actions that have been shown to affect the ability of the heart to protect itself against ischemia-reperfusion injury in preclinical models. However, the effects of these agents in doses used in the clinical setting to minimize ischemia-reperfusion injury and to affect clinical outcomes in patients with DM have yet to be shown. The clinical implications as well as the mechanisms of protection should be further studied.

Concise Review: Mesenchymal Stem Cells for Acute Lung Injury: Role of Paracrine Soluble Factors

Morbidity and mortality have declined only modestly in patients with clinical acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), despite extensive research into the pathophysiology. Current treatment remains primarily supportive with lung-protective ventilation and a fluid conservative strategy. Pharmacologic therapies that reduce the severity of lung injury in preclinical models have not yet been translated to effective clinical treatment options. Consequently, further research in translational therapies is needed. Cell-based therapy with mesenchymal stem cells (MSCs) is one attractive new therapeutic approach. MSCs have the capacity to secrete multiple paracrine factors that can regulate endothelial and epithelial permeability, decrease inflammation, enhance tissue repair, and inhibit bacterial growth. This review will focus on recent studies, which support the potential therapeutic use of MSCs in ALI/ARDS, with an emphasis on the role of paracrine soluble factors.

Hypercapnia and Acidosis in Sepsis

Acute respiratory distress syndrome is a devastating disease that causes substantial morbidity and mortality. Mechanical ventilation can worsen lung injury, whereas ventilatory strategies that reduce lung stretch, resulting in a "permissive" hypercapnic acidosis (HCA), improve outcome. HCA directly reduces nonsepsis-induced lung injury in preclinical models and, therefore, has therapeutic potential in these patients. These beneficial effects are mediated via inhibition of the host immune response, particularly cytokine signaling, phagocyte function, and the adaptive immune response. Of concern, these immunosuppressive effects of HCA may hinder the host response to microbial infection. Recent studies suggest that HCA is protective in the earlier phases of bacterial pneumonia-induced sepsis but may worsen injury in the setting of prolonged lung sepsis. In contrast, HCA is protective in preclinical models of early and prolonged systemic sepsis. Buffering of the HCA is not beneficial and may worsen pneumonia-induced injury.

Association between urokinase haplotypes and outcome from infection-associated acute lung injury

Alterations in coagulation, including elevated pulmonary and systemic concentrations of urokinase, are frequent in patients with acute lung injury (ALI). Urokinase potentiates neutrophil activation and contributes to the severity of pulmonary injury in preclinical models of ALI. The objective of this study was to examine associations between polymorphisms and haplotypes of urokinase with risk for and outcomes from ALI. Prospective cohorts of healthy European-American adults and those with infection-associated ALI. Academic medical centers participating in NIH funded studies of low tidal volume ventilation for ALI. Controls were 175 healthy European-American subjects. Patients were 252 individuals with infection-associated ALI, prospectively followed for 60 days for mortality. Genetic polymorphisms and haplotypes in the urokinase gene were determined. Six polymorphisms, rs1916341, rs2227562, rs2227564, rs2227566, rs2227571, and rs4065, defining 98% of all urokinase haplotypes, were analyzed. There were no statistically significant associations between any single urokinase polymorphism or haplotype and risk for developing ALI. In contrast, there was a statistically significant relationship between the CGCCCC haplotype and both 60-day mortality and ventilator-free days that remained present in a multivariate analysis controlling for age and sex (p=0.033 for 60-day mortality and <0.001 for ventilator-free days). These results identify a specific urokinase haplotype as a genetic risk factor for higher mortality and more severe clinical outcome in patients with infection-associated ALI.