Michael D. Goodman

Endothelial cell surface F1-FO ATP synthase is active in ATP synthesis and is inhibited by angiostatin

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F1-FO ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F1 ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F1 ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the α- and β-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

Regulating RISK: a role for JAK-STAT signaling in postconditioning?

Postconditioning (POC), a novel strategy of cardioprotection against ischemia-reperfusion injury, is clinically attractive because of its therapeutic application at the predictable onset of reperfusion. POC activates several intracellular kinase signaling pathways, including phosphatidylinositol 3-kinase (PI3K)-Akt (RISK). The regulation of POC-induced survival kinase signaling, however, has not been fully characterized. JAK-STAT activation is integral to cardiac ischemic tolerance and may provide upstream regulation of RISK. We hypothesized that POC requires the activation of both JAK-STAT and RISK signaling. Langendorff-perfused mouse hearts were subjected to 30 min of global ischemia and 40 min of reperfusion, with or without POC immediately after ischemia. A separate group of POC hearts was treated with AG 490, a JAK2 inhibitor, Stattic, a specific STAT3 inhibitor, or LY-294002, a PI3K inhibitor, at the onset of reperfusion. Cardiomyocyte-specific STAT3 knockout (KO) hearts were also subjected to non-POC or POC protocols. Myocardial performance (+dP/dt(max), mmHg/s) was assessed throughout each perfusion protocol. Phosphorylated (p-) STAT3 and Akt expression was analyzed by Western immunoblotting. POC enhanced myocardial functional recovery and increased expression of p-STAT3 and p-Akt. JAK-STAT inhibition abrogated POC-induced functional protection. STAT3 inhibition decreased expression of both p-STAT3 and p-Akt. PI3K inhibition also attenuated POC-induced cardioprotection and reduced p-Akt expression but had no effect on STAT3 phosphorylation. Interestingly, STAT3 KO hearts undergoing POC exhibited improved ischemic tolerance compared with KO non-POC hearts. POC induces myocardial functional protection by activating the RISK pathway. JAK-STAT signaling, however, is insufficient for effective POC without PI3K-Akt activation.

A murine model of mild traumatic brain injury exhibiting cognitive and motor deficits

BACKGROUND Mild traumatic brain injury (TBI) is a serious public health concern affecting more than 1.7 million people in the United States annually. Mild TBI is difficult to diagnose and is clinically associated with impaired motor coordination and cognition. METHODS We subjected mice to a mild TBI (mTBI-1 or mTBI-2) induced by a weight drop model. We assessed brain injury histologically and biochemically, the latter by serum neuron-specific enolase and glial fibrillary acidic protein. Systemic and brain inflammation were measured by cytokine array. We determined blood-brain barrier integrity by cerebral vascular leakage of micromolecular and macromolecular fluorescent molecules. We evaluated mice using a rotarod device and novel object recognition to measure motor coordination and cognition, respectively. RESULTS Mice undergoing mTBI-1 or mTBI-2 had significant deficits in motor coordination and cognition for several days after injury compared with controls. Furthermore, both mTBI-1 and mTBI-2 caused micromolecular leakage in the blood-brain barrier, whereas only mTBI-2 caused macromolecular leakage. Serum neuron-specific enolase and glial fibrillary acidic protein were elevated acutely and corresponded to the degree of injury, but returned to baseline within 24 h. Serum cytokines interleukin-6 and keratinocyte-derived chemokine were significantly increased within 90 min of TBI. Interleukin-6 levels correlated with the degree of injury. CONCLUSIONS The current study provides a reproducible model of mild TBI in mice that exhibits pathologic features of mild TBI in humans. Furthermore, our data suggest that serum cytokines, such as IL-6, may be effective biomarkers for severity of head injury.

Interleukin 6 mediates neuroinflammation and motor coordination deficits after mild traumatic brain injury and brief hypoxia in mice.

ABSTRACT Traumatic brain injury (TBI) is a leading cause of mortality and disability. Acute postinjury insults after TBI, such as hypoxia, contribute to secondary brain injury and worse clinical outcomes. The functional and neuroinflammatory effects of brief episodes of hypoxia experienced following TBI have not been evaluated. Our previous studies have identified interleukin 6 (IL-6) as a potential mediator of mild TBI–induced pathology. In the present study, we sought to determine the effects of brief hypoxia on mild TBI and whether IL-6 played a role in the neuroinflammatory and functional deficits after injury. A murine model of mild TBI was induced by a weight drop (500 g from 1.5 cm). After injury, mice were exposed to immediate hypoxia (FIO2 = 15.1%) or normoxia (FIO2 = 21%) for 30 min. Serum and brain samples were analyzed for inflammatory cytokines 24 h after TBI. Neuron-specific enolase was measured as a serum biomarker of brain injury. Evaluation of motor coordination was performed for 5 days after TBI using a rotarod device. In some animals, anti–IL-6 was administered following TBI and hypoxia to neutralize systemic IL-6. Mice undergoing TBI had significant increases in brain injury. Exposure to brief hypoxia after TBI resulted in a more than 5-fold increase in serum neuron-specific enolase. This increase was associated with increases in serum and brain cytokine expression, suggesting that brief hypoxia exacerbates systemic and brain inflammation. Neutralization of IL-6 suppressed postinjury neuroinflammation and neuronal injury. In addition, TBI and hypoxia induced significant motor coordination deficits that were completely abrogated by IL-6 blockade. Exposure to hypoxia after TBI induces neuroinflammation and brain injury. These changes can be mitigated by neutralization of systemic IL-6. Interleukin 6 blockade also corrected the TBI-induced deficit in motor coordination. These data suggest that systemic IL-6 modulates the degree of neuroinflammation and contributes to reduced motor coordination after mild TBI.

Resuscitation with fresh whole blood ameliorates the inflammatory response after hemorrhagic shock.

BACKGROUND Hemorrhagic shock is the leading cause of potentially preventable death after traumatic injury. Hemorrhage and subsequent resuscitation may result in a dysfunctional systemic inflammatory response and multisystem organ failure, leading to delayed mortality. Clinical evidence supports improved survival and reduced morbidity when fresh blood products are used as resuscitation strategies. We hypothesized that the transfusion of fresh whole blood (FWB) attenuates systemic inflammation and reduces organ injury when compared with conventional crystalloid resuscitation after hemorrhagic shock. METHODS Male mice underwent femoral artery cannulation and hemorrhage to a systolic blood pressure of 25 mm Hg +/- 5 mm Hg. After 60 minutes, the mice were resuscitated with either FWB or lactated Ringers solution (LR). Mice were decannulated and killed at intervals for tissue histology, serum cytokine analysis, and vascular permeability studies. Separate groups of mice were followed for survival studies. RESULTS When compared with FWB, mice resuscitated with LR required increased resuscitation fluid volume to reach goal systolic blood pressure. When compared with sham or FWB-resuscitated mice, LR resuscitation resulted in increased serum cytokine levels of macrophage inflammatory protein-1alpha, interleukin-6, interleukin-10, macrophage-derived chemokine, KC, and granulocyte macrophage colony stimulating factor as well as increased lung injury and pulmonary capillary permeability. No survival differences were seen between animals resuscitated with LR or FWB. CONCLUSIONS Resuscitation with LR results in increased systemic inflammation, vascular permeability, and lung injury after hemorrhagic shock. Resuscitation with FWB attenuates the inflammation and lung injury seen with crystalloid resuscitation. These findings suggest that resuscitation strategies using fresh blood products potentially reduce systemic inflammation and organ injury after hemorrhagic shock.

Hypobaric hypoxia exacerbates the neuroinflammatory response to traumatic brain injury.

OBJECTIVE To determine the inflammatory effects of time-dependent exposure to the hypobaric environment of simulated aeromedical evacuation following traumatic brain injury (TBI). METHODS Mice were subjected to a blunt TBI or sham injury. Righting reflex response (RRR) time was assessed as an indicator of neurologic recovery. Three or 24 h (Early and Delayed groups, respectively) after TBI, mice were exposed to hypobaric flight conditions (Fly) or ground-level control (No Fly) for 5 h. Arterial blood gas samples were obtained from all groups during simulated flight. Serum and cortical brain samples were analyzed for inflammatory cytokines after flight. Neuron specific enolase (NSE) was measured as a serum biomarker of TBI severity. RESULTS TBI resulted in prolonged RRR time compared with sham injury. After TBI alone, serum levels of interleukin-6 (IL-6) and keratinocyte-derived chemokine (KC) were increased by 6 h post-injury. Simulated flight significantly reduced arterial oxygen saturation levels in the Fly group. Post-injury altitude exposure increased cerebral levels of IL-6 and macrophage inflammatory protein-1α (MIP-1α), as well as serum NSE in the Early but not Delayed Flight group compared to ground-level controls. CONCLUSIONS The hypobaric environment of aeromedical evacuation results in significant hypoxia. Early, but not delayed, exposure to a hypobaric environment following TBI increases the neuroinflammatory response to injury and the severity of secondary brain injury. Optimization of the post-injury time to fly using serum cytokine and biomarker levels may reduce the potential secondary cerebral injury induced by aeromedical evacuation.

Preinjury alcohol exposure attenuates the neuroinflammatory response to traumatic brain injury

BACKGROUND Traumatic brain injury (TBI) initiates a neuroinflammatory response that increases the risk of TBI-related mortality. Acute alcohol intoxication at the time of TBI is associated with improved survival. Ethanol is recognized as a systemic immunomodulator that may also impart neuroprotection. The effects of alcohol on TBI-induced neuroinflammation, however, are unknown. We hypothesized that ethanol treatment prior to TBI may provide neuroprotection by diminishing the neuroinflammatory response to injury. MATERIALS AND METHODS Mice underwent gavage with ethanol (EtOH) or water (H2O) prior to TBI. Animals were subjected to blunt TBI or sham injury (Sham). Posttraumatic rapid righting reflex (RRR) and apnea times were assessed. Cerebral and serum samples were analyzed by ELISA for inflammatory cytokine levels. Serum neuron-specific enolase (NSE), a biomarker of injury severity, was also measured. RESULTS Neurologic recovery from TBI was more rapid in H2O-treated mice compared with EtOH-treated mice. However, EtOH/TBI mice had a 4-fold increase in RRR time compared with EtOH/Sham, whereas H2O/TBI mice had a 15-fold increase in RRR time compared with H2O/Sham. Ethanol intoxication at the time of TBI significantly increased posttraumatic apnea time. Preinjury EtOH treatment was associated with reduced levels of proinflammatory cytokines IL-6, KC, MCP-1, and MIP-1α post TBI. NSE was significantly increased post injury in the H2O/TBI group compared with H2O/Sham but was not significantly reduced by EtOH pretreatment. CONCLUSIONS Alcohol treatment prior to TBI reduces the local neuroinflammatory response to injury. The decreased neurologic and inflammatory impact of TBI in acutely intoxicated patients may be responsible for improved clinical outcomes.

Murine blood banking: characterization and comparisons to human blood.

Blood transfusion remains an essential treatment of acute anemia. Current storage processes allow the efficient administration of blood products. Erythrocytes undergo morphological and biochemical changes during storage that may affect outcomes after transfusion. A reliable small-animal model would be ideal to examine the effects of stored blood products after transfusion. The objective of this study was to characterize the storage of murine erythrocytes for future application to animal models of acute anemia. Blood samples were collected from male mice and human volunteers, separated into components, and stored. At intervals, morphological and biochemical analysis was performed. Lactate, potassium, hemoglobin, and hemolysis were determined, and cell morphology was evaluated with light microscopy. Murine packed red blood cells (pRBCs) aged more rapidly than human samples. Murine pRBCs exhibited higher lactate levels (34.9 ± 1.3 mmol/L vs. 18.1 ± 1.0 mmol/L, mouse vs. human) and more severe acidosis as indicated by pH (6.56 ± 0.02 vs. 6.79 ± 0.04, mouse vs. human). Murine pRBCs hemolyzed earlier (11.2 ± 3.7 g vs. 0.7 ± 0.3 g, mouse vs. human after 21 days of storage) and more rapidly than human pRBCs. Corpuscular changes consistent with red cell storage lesions appeared earlier in murine samples compared with human stored pRBCs. Compared with human pRBCs, murine pRBCs exhibit similar but more accelerated aging processes under standard storage conditions. Characterization of the murine red cell storage lesion will allow the application of stored blood components to future investigations into the treatment of acute anemia in experimental murine models.

Video-assisted thoracoscopic surgery for acute thoracic trauma.

Background: Operative intervention for thoracic trauma typically requires thoracotomy. We hypothesized that thoracoscopy may be safely and effectively utilized for the acute management of thoracic injuries. Materials and Methods: The Trauma Registry of a Level I trauma center was queried from 1999 through 2010 for all video-assisted thoracic procedures within 24 h of admission. Data collected included initial vital signs, operative indication, intraoperative course, and postoperative outcome. Results: Twenty-three patients met inclusion criteria: 3 (13%) following blunt injury and 20 (87%) after penetrating trauma. Indications for urgent thoracoscopy included diaphragmatic/esophageal injury, retained hemothorax, ongoing hemorrhage, and open/persistent pneumothorax. No conversions to thoracotomy were required and no patient required re-operation. Mean postoperative chest tube duration was 2.9 days and mean length of stay was 5.6 days. Conclusion: Video-assisted thoracoscopic surgery is safe and effective for managing thoracic trauma in hemodynamically stable patients within the first 24 h post-injury.

Damage control resuscitation decreases systemic inflammation after hemorrhage.

BACKGROUND Severe hemorrhagic shock and resuscitation initiates a dysfunctional systemic inflammatory response leading to end-organ injury. Clinical evidence supports the transfusion of high ratios of plasma and packed red blood cells (pRBCs) in the treatment of hemorrhagic shock. The effects of resuscitation with different ratios of fresh blood products on inflammation and organ injury have not yet been characterized. MATERIALS AND METHODS Mice underwent femoral artery cannulation and pressure-controlled hemorrhage for 60 min, then resuscitation with fresh plasma and pRBCs collected from donor mice. Plasma alone, pRBCs alone, and ratios of 2:1, 1:1, and 1:2 plasma:pRBCs were used for resuscitation strategies. Mice were sacrificed to determine biochemical and hematologic parameters, serum cytokine concentrations, tissue myeloperoxidase levels, and vascular permeability. RESULTS Compared with other resuscitation strategies, mice resuscitated with pRBCs alone exhibited increased hemoglobin levels, while other hematologic and biochemical parameters were not significantly different among groups. Compared with 1:1, mice resuscitated with varying ratios of plasma:pRBCs exhibited increased cytokine concentrations of KC, MIP-1α, and MIP-2, and increased intestinal and lung myeloperoxidase levels. Mice resuscitated with 1:1 had decreased vascular permeability in the intestine and lung as compared with other groups. CONCLUSIONS Resuscitation with a 1:1 ratio of fresh plasma:pRBCs results in decreased systemic inflammation and attenuated organ injury. These findings support the potential advantage of transfusing blood products in physiologic ratios to improve the treatment of severe hemorrhagic shock.