1,720,982 research outputs found
Effect of Remote Ischemic Conditioning on Lipopolysaccharide-induced Pulmonary Inflammation
No full textRemote ischemic preconditioning (RIPC), an intervention whereby an extremity undergoes brief repeated cycles of ischemia/reperfusion (I/R), has been shown to exert protective effects on distant organ I/R injury. Its potential benefit in organ injury induced by other inflammatory stimuli has not been determined. To test whether RIPC would exert protective effects on LPS-induced pulmonary inflammation, mice were subjected to hindlimb RIPC using a tourniquet prior to intratracheal LPS. RIPC significantly reduced leukocyte recruitment and protein leakage in the lungs at 4 hours after LPS. Lung ICAM-1 mRNA expression was also reduced, whereas TNF-ι and IL-1β were upregulated, although these changes were statistically insignificant. However, the protective effect of RIPC was lost by 24 hours after LPS. Moreover, delayed conditioning and dual conditioning before and after LPS administration did not prevent lung inflammation. In conclusion, RIPC provides early protection against LPS-induced pulmonary inflammation in mice, but the effect is not sustained.M.Sc
Effect of Remote Ischemic Conditioning on Lipopolysaccharide-induced Pulmonary Inflammation
Full text linkRemote ischemic preconditioning (RIPC), an intervention whereby an extremity undergoes brief repeated cycles of ischemia/reperfusion (I/R), has been shown to exert protective effects on distant organ I/R injury. Its potential benefit in organ injury induced by other inflammatory stimuli has not been determined. To test whether RIPC would exert protective effects on LPS-induced pulmonary inflammation, mice were subjected to hindlimb RIPC using a tourniquet prior to intratracheal LPS. RIPC significantly reduced leukocyte recruitment and protein leakage in the lungs at 4 hours after LPS. Lung ICAM-1 mRNA expression was also reduced, whereas TNF-ι and IL-1β were upregulated, although these changes were statistically insignificant. However, the protective effect of RIPC was lost by 24 hours after LPS. Moreover, delayed conditioning and dual conditioning before and after LPS administration did not prevent lung inflammation. In conclusion, RIPC provides early protection against LPS-induced pulmonary inflammation in mice, but the effect is not sustained.M.Sc
Cellular Mechanisms of the Systemic Inflammatory Response Following Resuscitated Hemorrhagic Shock: The Role of Reactive Oxygen Species and Toll-like Receptor 4
Full text linkAcute Respiratory Distress Syndrome (ARDS) following hemorrhagic
shock/resuscitation (S/R) is an important contributor to late morbidity and mortality in trauma
patients. S/R promotes ARDS by inducing oxidative stress that primes cells of the innate
immune system for excessive responsiveness to small inflammatory stimuli, termed the “twohit”
hypothesis. Activated alveolar macrophages (AM) play a central role and when recovered
from S/R animals exhibit an exaggerated responsiveness to lipopolysaccharide (LPS) with
increased activation of the proinflammatory transcription factor NF-κB, and augmented
expression of cytokines. LPS triggers AM signalling through Toll like receptor 4 (TLR4), which
resides in plasma membrane lipid rafts.
The objective of this work is to define cellular mechanisms of macrophage priming by
oxidative stress following shock resuscitation. The main hypothesis investigated is that altered
cellular distribution of TLR4 can lead to macrophage priming and antioxidant resuscitation
strategies can diminish these effects.
AM of rodents, exposed in vivo to oxidant stress following S/R, increase their surface
levels of TLR4, which in turn results in augmented NF-κB translocation in response to small
doses of LPS. Furthermore, in vitro H2O2 treatment of RAW 264.7 macrophages results in
similar TLR4 surface translocation. Depletion of intracellular calcium, disruption of the
cytoskeleton or inhibition of the Src kinases prevents the H2O2-induced TLR4 translocation,
suggesting the involvement of receptor exocytosis. Further, fluorescent resonance energy
iii
transfer between TLR4 and lipid rafts as well as biochemical raft analysis demonstrated that
oxidative stress redistributes TLR4 to surface lipid rafts. Preventing the oxidant-induced
movement of TLR4 to lipid rafts using methyl-ß-cyclodextrin precluded the increased
responsiveness of cells to LPS after H2O2 treatment. Further, AM priming by oxidative stress
can be diminished by early exposure to resuscitation regimens with direct or indirect systemic
antioxidant effects, such as 25% albumin, N-acetylcysteine and hypertonic saline.
Hyperosmolarity was found to modulate AM TLR4 gene and protein expression.
Collectively, these studies suggest a novel mechanism whereby oxidative stress might
prime the responsiveness of cells of the innate immune system. Targeting the TLR4 signalling
pathway early during shock resuscitation may represent an anti-inflammatory strategy able to
ameliorate late morbidity and mortality following S/R.Ph
The Role of Ceramide in Oxidant-mediated Priming of Macrophages for LPS Signaling
Full text linkIntroduction: Civilian trauma remains a significant health care problem in North American society. Hemorrhagic shock and resuscitation (S/R) have been shown to prime the immune system for an exaggerated response to subsequent otherwise innocuous inflammatory stimuli such as lipopolysaccharide (LPS), resulting in multiple organ failure or death. Using a rodent model of lung injury, we previously demonstrated that antecedent S/R leads to augmented LPS-induced lung injury by way of heightened NF-κB nuclear translocation, resulting in increased elaboration of pro-inflammatory cytokines in alveolar macrophages. Further studies revealed that oxidative stress generated during S/R is responsible for this priming phenomenon. Our group recently identified two significant alterations to LPS signaling under oxidative stress conditions in macrophages: 1) the rapid recruitment of the LPS receptor Toll-like receptor 4 (TLR4) to membrane lipid rafts, and 2) the reprogramming of LPS signaling to a Src-dependent pathway involving phosphatidylinositol 3-kinase (PI3K).
Major Objective and Hypothesis: The objective of this thesis is to elucidate the molecular mechanisms underlying the augmented cellular responsiveness observed in macrophages following oxidative stress. The central hypothesis is that oxidative stress regulates LPS signaling by altering the activation and assembly of TLR4 receptor signaling components through generation of the lipid ceramide.
Summary of Findings: In the first paper, we demonstrate that the antioxidant stilbazulenyl nitrone (STAZN), a novel second-generation azulenyl nitrone, is protective in a rodent two-hit model of lung injury involving hemorrhagic S/R and subsequent intra-tracheal LPS injection. Resultant oxidative stress and lung injury in vivo were significantly reduced by STAZN following S/R and LPS. In the second paper, we explore the mechanism underlying oxidant-induced surface up-regulation of TLR4 in macrophages. Using immunofluorescence microscopy and flow cytometry techniques, hydrogen peroxide in vitro and hemorrhagic S/R in vivo are shown to induce TLR4 translocation in macrophages in a ceramide and Src-dependent manner, and the enzyme acid sphingomyelinase (ASM) is shown to mediate ceramide generation. In the third paper, the role of ceramide in oxidant-induced macrophage priming for LPS signaling is investigated. Ceramide generation via ASM is shown to have a prominent upstream role in oxidant activation of the PI3K/Akt pathway via Src kinases in macrophages. Furthermore, oxidative stress is shown to reprogram LPS signaling to a ceramide dependent pathway.
Conclusion: Together, these findings highlight the role of oxidative stress in mediating augmented cellular responsiveness following S/R, and describe the role of ceramide as a central upstream mediator of oxidant priming in macrophages. The hierarchy of signaling molecules and interactions described herein represent novel targets for modulating oxidative stress in the treatment of critical illness and organ injury.Ph
Cellular Mechanisms of the Systemic Inflammatory Response Following Resuscitated Hemorrhagic Shock: The Role of Reactive Oxygen Species and Toll-like Receptor 4
No full textAcute Respiratory Distress Syndrome (ARDS) following hemorrhagic
shock/resuscitation (S/R) is an important contributor to late morbidity and mortality in trauma
patients. S/R promotes ARDS by inducing oxidative stress that primes cells of the innate
immune system for excessive responsiveness to small inflammatory stimuli, termed the “twohit”
hypothesis. Activated alveolar macrophages (AM) play a central role and when recovered
from S/R animals exhibit an exaggerated responsiveness to lipopolysaccharide (LPS) with
increased activation of the proinflammatory transcription factor NF-κB, and augmented
expression of cytokines. LPS triggers AM signalling through Toll like receptor 4 (TLR4), which
resides in plasma membrane lipid rafts.
The objective of this work is to define cellular mechanisms of macrophage priming by
oxidative stress following shock resuscitation. The main hypothesis investigated is that altered
cellular distribution of TLR4 can lead to macrophage priming and antioxidant resuscitation
strategies can diminish these effects.
AM of rodents, exposed in vivo to oxidant stress following S/R, increase their surface
levels of TLR4, which in turn results in augmented NF-κB translocation in response to small
doses of LPS. Furthermore, in vitro H2O2 treatment of RAW 264.7 macrophages results in
similar TLR4 surface translocation. Depletion of intracellular calcium, disruption of the
cytoskeleton or inhibition of the Src kinases prevents the H2O2-induced TLR4 translocation,
suggesting the involvement of receptor exocytosis. Further, fluorescent resonance energy
iii
transfer between TLR4 and lipid rafts as well as biochemical raft analysis demonstrated that
oxidative stress redistributes TLR4 to surface lipid rafts. Preventing the oxidant-induced
movement of TLR4 to lipid rafts using methyl-ß-cyclodextrin precluded the increased
responsiveness of cells to LPS after H2O2 treatment. Further, AM priming by oxidative stress
can be diminished by early exposure to resuscitation regimens with direct or indirect systemic
antioxidant effects, such as 25% albumin, N-acetylcysteine and hypertonic saline.
Hyperosmolarity was found to modulate AM TLR4 gene and protein expression.
Collectively, these studies suggest a novel mechanism whereby oxidative stress might
prime the responsiveness of cells of the innate immune system. Targeting the TLR4 signalling
pathway early during shock resuscitation may represent an anti-inflammatory strategy able to
ameliorate late morbidity and mortality following S/R.Ph
The Role of Ceramide in Oxidant-mediated Priming of Macrophages for LPS Signaling
No full textIntroduction: Civilian trauma remains a significant health care problem in North American society. Hemorrhagic shock and resuscitation (S/R) have been shown to prime the immune system for an exaggerated response to subsequent otherwise innocuous inflammatory stimuli such as lipopolysaccharide (LPS), resulting in multiple organ failure or death. Using a rodent model of lung injury, we previously demonstrated that antecedent S/R leads to augmented LPS-induced lung injury by way of heightened NF-κB nuclear translocation, resulting in increased elaboration of pro-inflammatory cytokines in alveolar macrophages. Further studies revealed that oxidative stress generated during S/R is responsible for this priming phenomenon. Our group recently identified two significant alterations to LPS signaling under oxidative stress conditions in macrophages: 1) the rapid recruitment of the LPS receptor Toll-like receptor 4 (TLR4) to membrane lipid rafts, and 2) the reprogramming of LPS signaling to a Src-dependent pathway involving phosphatidylinositol 3-kinase (PI3K).
Major Objective and Hypothesis: The objective of this thesis is to elucidate the molecular mechanisms underlying the augmented cellular responsiveness observed in macrophages following oxidative stress. The central hypothesis is that oxidative stress regulates LPS signaling by altering the activation and assembly of TLR4 receptor signaling components through generation of the lipid ceramide.
Summary of Findings: In the first paper, we demonstrate that the antioxidant stilbazulenyl nitrone (STAZN), a novel second-generation azulenyl nitrone, is protective in a rodent two-hit model of lung injury involving hemorrhagic S/R and subsequent intra-tracheal LPS injection. Resultant oxidative stress and lung injury in vivo were significantly reduced by STAZN following S/R and LPS. In the second paper, we explore the mechanism underlying oxidant-induced surface up-regulation of TLR4 in macrophages. Using immunofluorescence microscopy and flow cytometry techniques, hydrogen peroxide in vitro and hemorrhagic S/R in vivo are shown to induce TLR4 translocation in macrophages in a ceramide and Src-dependent manner, and the enzyme acid sphingomyelinase (ASM) is shown to mediate ceramide generation. In the third paper, the role of ceramide in oxidant-induced macrophage priming for LPS signaling is investigated. Ceramide generation via ASM is shown to have a prominent upstream role in oxidant activation of the PI3K/Akt pathway via Src kinases in macrophages. Furthermore, oxidative stress is shown to reprogram LPS signaling to a ceramide dependent pathway.
Conclusion: Together, these findings highlight the role of oxidative stress in mediating augmented cellular responsiveness following S/R, and describe the role of ceramide as a central upstream mediator of oxidant priming in macrophages. The hierarchy of signaling molecules and interactions described herein represent novel targets for modulating oxidative stress in the treatment of critical illness and organ injury.Ph
Novel Imaging-Based Biomarkers for Identifying Carotid Plaque Vulnerability
Full text linkCarotid artery disease has traditionally been assessed based on the degree of luminal narrowing. However, this approach, which solely relies on carotid stenosis, is currently being questioned with regard to modern risk stratification approaches. Recent guidelines have introduced the concept of the “vulnerable plaque,” emphasizing specific features such as thin fibrous caps, large lipid cores, intraplaque hemorrhage, plaque rupture, macrophage infiltration, and neovascularization. In this context, imaging-based biomarkers have emerged as valuable tools for identifying higher-risk patients. Non-invasive imaging modalities and intravascular techniques, including ultrasound, computed tomography, magnetic resonance imaging, intravascular ultrasound, optical coherence tomography, and near-infrared spectroscopy, have played pivotal roles in characterizing and detecting unstable carotid plaques. The aim of this review is to provide an overview of the evolving understanding of carotid artery disease and highlight the significance of imaging techniques in assessing plaque vulnerability and informing clinical decision-making
Effect of Remote Ischemic Conditioning in Hemorrhagic Shock/Resuscitation
No full textTraumatic injuries remain a leading cause of death worldwide despite advances in resuscitation care, causing substantial burden to society. Trauma complicated with hemorrhagic shock/resuscitation (S/R) represents a form of global ischemia/reperfusion injury that promotes oxidative stress, systemic inflammation, and coagulopathy, which together contribute to organ dysfunction and poor outcomes. Therefore, strategies directed at preventing onset of ischemia/reperfusion injury have substantial potential to improve outcomes. Remote ischemic conditioning (RIC) is a simple non-invasive intervention in which a limb is subjected to sequential cycles of brief ischemia/reperfusion by inflation and deflation of a pressure cuff. RIC protects organs against ischemia/reperfusion injury in both experimental and clinical settings, but its effects in S/R has not been well defined. In this thesis, we investigated the efficacy of RIC in both pre-clinical and clinical settings. We first demonstrated in a murine model of S/R that RIC prevented organ injury and inflammation when administered before shock, during shock, and at resuscitation, suggesting its potential utility in trauma. This protective effect was mediated by induction of antioxidant response through the transcription factor Nrf2. Furthermore, humoral factors liberated by RIC prevented neutrophil migration in response to injury and oxidant-induced mortality. A unique finding was that transfusion with RIC donor blood protected the recipient animal from S/R induced organ injury and inflammation, suggesting a novel paradigm to administer RIC. To translate our findings to the clinical setting, we conducted a Phase II randomized controlled trial to investigate the feasibility and effects on the immune-inflammatory and coagulation profiles of RIC administered to trauma patients sustaining hemorrhagic shock. A total of 50 patients were enrolled, in whom in-hospital RIC was successfully and safely administered to most of the patients. Although RIC did not significantly influence clinical outcomes, RIC prevented neutrophil degranulation, modulated release of Th2 chemokines, and exerted a favourable coagulation profile in the early resuscitation period. Taken together, our work establishes RIC as a potential therapeutic intervention that can be safely administered to patients in the acute injury setting. Future studies aimed at characterizing the mechanisms of RIC and optimizing its administration may lead to beneficial outcomes in trauma patients.Ph.D
Effect of Remote Ischemic Conditioning in Hemorrhagic Shock/Resuscitation
No full textTraumatic injuries remain a leading cause of death worldwide despite advances in resuscitation care, causing substantial burden to society. Trauma complicated with hemorrhagic shock/resuscitation (S/R) represents a form of global ischemia/reperfusion injury that promotes oxidative stress, systemic inflammation, and coagulopathy, which together contribute to organ dysfunction and poor outcomes. Therefore, strategies directed at preventing onset of ischemia/reperfusion injury have substantial potential to improve outcomes. Remote ischemic conditioning (RIC) is a simple non-invasive intervention in which a limb is subjected to sequential cycles of brief ischemia/reperfusion by inflation and deflation of a pressure cuff. RIC protects organs against ischemia/reperfusion injury in both experimental and clinical settings, but its effects in S/R has not been well defined. In this thesis, we investigated the efficacy of RIC in both pre-clinical and clinical settings. We first demonstrated in a murine model of S/R that RIC prevented organ injury and inflammation when administered before shock, during shock, and at resuscitation, suggesting its potential utility in trauma. This protective effect was mediated by induction of antioxidant response through the transcription factor Nrf2. Furthermore, humoral factors liberated by RIC prevented neutrophil migration in response to injury and oxidant-induced mortality. A unique finding was that transfusion with RIC donor blood protected the recipient animal from S/R induced organ injury and inflammation, suggesting a novel paradigm to administer RIC. To translate our findings to the clinical setting, we conducted a Phase II randomized controlled trial to investigate the feasibility and effects on the immune-inflammatory and coagulation profiles of RIC administered to trauma patients sustaining hemorrhagic shock. A total of 50 patients were enrolled, in whom in-hospital RIC was successfully and safely administered to most of the patients. Although RIC did not significantly influence clinical outcomes, RIC prevented neutrophil degranulation, modulated release of Th2 chemokines, and exerted a favourable coagulation profile in the early resuscitation period. Taken together, our work establishes RIC as a potential therapeutic intervention that can be safely administered to patients in the acute injury setting. Future studies aimed at characterizing the mechanisms of RIC and optimizing its administration may lead to beneficial outcomes in trauma patients.Ph.D
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