"Mechanisms of nitric oxide synthase uncoupling in endotoxin-induced acute lung injury: role of asymmetric dimethylarginine" appeared in a 2010 issue of Vascular Pharmacology and summarized Stephen M. Black's research into acute lung injury/sepsis.
This study investigated whether supplementing glutathione (GSH) could attenuate lipopolysaccharide (LPS)-induced mitochondrial dysfunction and apoptosis in a mouse model of acute lung injury (ALI). The researchers found that LPS increased oxidative stress, mitochondrial dysfunction, and apoptosis in mouse lung tissue. However, pre-treating the mice with GSH-ethyl ester prevented the LPS-induced increases in oxidative stress, preserved mitochondrial function, and reduced apoptosis. Thus, GSH supplementation may protect against LPS-induced mitochondrial damage and cellular apoptosis in ALI.
This document summarizes a study that investigated the determinants of oxygen and carbon dioxide transfer during extracorporeal membrane oxygenation (ECMO) in an experimental pig model of multiple organ dysfunction syndrome (MODS). The study found that oxygen transfer was positively associated with blood flow and negatively associated with pre-membrane oxygen saturation and carbon dioxide levels, while carbon dioxide transfer was positively associated with blood and gas flows and pre-membrane carbon dioxide levels. Passing blood through the ECMO circuit increased pH and decreased carbon dioxide levels. Both oxygen and carbon dioxide transfers were significantly determined by blood flow.
- 60 apoE-/- mice were exposed to either clean filtered air, concentrated ambient particles (CAPs), 200 ppb ozone (O3), or both CAPs and O3 for 5 hours/day, 4 days/week for 8 weeks.
- Mice exposed to CAPs and CAPs with O3 showed the greatest increases in systolic and diastolic blood pressure compared to the air control group. The blood pressure increase appeared to be driven by PM exposure rather than the combined exposure to O3.
- Exposure to CAPs significantly raised blood pressure in the atherosclerosis-susceptible mice, while O3 exposure alone had a lesser effect. This highlights the importance of PM exposure in
1) The document discusses high-frequency oscillatory ventilation (HFOV), a lung-protective ventilation strategy that uses small, high-frequency breaths to ventilate patients with acute respiratory distress syndrome (ARDS).
2) Two randomized controlled trials (MOAT and Bollen) compared HFOV to conventional ventilation in adults with ARDS but found no significant differences in mortality between the groups.
3) The trials were underpowered and had some limitations in their ventilation protocols, so more research is still needed to determine if HFOV provides benefits over lung-protective conventional ventilation for adults with ARDS.
This study investigated the mechanism by which salicylates (aspirin and sodium salicylate) dilate blood vessels. The researchers found that salicylates relaxed contractions induced by most constrictors through inhibiting RhoA/Rho-kinase-mediated calcium sensitization. Salicylates inhibited PYK2-mediated activation of RhoA and Rho-kinase, as evidenced by decreased RhoA translocation and MYPT1 phosphorylation. Overexpression of PYK2 induced RhoA translocation and MYPT1 phosphorylation, effects that were inhibited by sodium salicylate. Consistent with these findings, sodium salicylate decreased blood pressure in spontaneous hypertensive rats but not in normotensive rats. The
Senior Research Presentation - Use of CO to treat ischemia reperfusion injuryjamickle
Carbon monoxide (CO) therapy shows promise in treating ischemia reperfusion injury (IRI) by reducing inflammation and cell death. Studies on rat models of stroke and organ transplantation found that low doses of inhaled CO during reperfusion improved tissue recovery and survival rates compared to no treatment. The mechanisms are not fully understood but may involve upregulation of the heme oxygenase system, which naturally produces CO. While results are promising, more research is needed to understand the mechanisms, test higher doses safely, and evaluate long term effects before clinical trials in humans.
This study investigated whether supplementing glutathione (GSH) could attenuate lipopolysaccharide (LPS)-induced mitochondrial dysfunction and apoptosis in a mouse model of acute lung injury (ALI). The researchers found that LPS increased oxidative stress, mitochondrial dysfunction, and apoptosis in mouse lung tissue. However, pre-treating the mice with GSH-ethyl ester prevented the LPS-induced increases in oxidative stress, preserved mitochondrial function, and reduced apoptosis. Thus, GSH supplementation may protect against LPS-induced mitochondrial damage and cellular apoptosis in ALI.
This document summarizes a study that investigated the determinants of oxygen and carbon dioxide transfer during extracorporeal membrane oxygenation (ECMO) in an experimental pig model of multiple organ dysfunction syndrome (MODS). The study found that oxygen transfer was positively associated with blood flow and negatively associated with pre-membrane oxygen saturation and carbon dioxide levels, while carbon dioxide transfer was positively associated with blood and gas flows and pre-membrane carbon dioxide levels. Passing blood through the ECMO circuit increased pH and decreased carbon dioxide levels. Both oxygen and carbon dioxide transfers were significantly determined by blood flow.
- 60 apoE-/- mice were exposed to either clean filtered air, concentrated ambient particles (CAPs), 200 ppb ozone (O3), or both CAPs and O3 for 5 hours/day, 4 days/week for 8 weeks.
- Mice exposed to CAPs and CAPs with O3 showed the greatest increases in systolic and diastolic blood pressure compared to the air control group. The blood pressure increase appeared to be driven by PM exposure rather than the combined exposure to O3.
- Exposure to CAPs significantly raised blood pressure in the atherosclerosis-susceptible mice, while O3 exposure alone had a lesser effect. This highlights the importance of PM exposure in
1) The document discusses high-frequency oscillatory ventilation (HFOV), a lung-protective ventilation strategy that uses small, high-frequency breaths to ventilate patients with acute respiratory distress syndrome (ARDS).
2) Two randomized controlled trials (MOAT and Bollen) compared HFOV to conventional ventilation in adults with ARDS but found no significant differences in mortality between the groups.
3) The trials were underpowered and had some limitations in their ventilation protocols, so more research is still needed to determine if HFOV provides benefits over lung-protective conventional ventilation for adults with ARDS.
This study investigated the mechanism by which salicylates (aspirin and sodium salicylate) dilate blood vessels. The researchers found that salicylates relaxed contractions induced by most constrictors through inhibiting RhoA/Rho-kinase-mediated calcium sensitization. Salicylates inhibited PYK2-mediated activation of RhoA and Rho-kinase, as evidenced by decreased RhoA translocation and MYPT1 phosphorylation. Overexpression of PYK2 induced RhoA translocation and MYPT1 phosphorylation, effects that were inhibited by sodium salicylate. Consistent with these findings, sodium salicylate decreased blood pressure in spontaneous hypertensive rats but not in normotensive rats. The
Senior Research Presentation - Use of CO to treat ischemia reperfusion injuryjamickle
Carbon monoxide (CO) therapy shows promise in treating ischemia reperfusion injury (IRI) by reducing inflammation and cell death. Studies on rat models of stroke and organ transplantation found that low doses of inhaled CO during reperfusion improved tissue recovery and survival rates compared to no treatment. The mechanisms are not fully understood but may involve upregulation of the heme oxygenase system, which naturally produces CO. While results are promising, more research is needed to understand the mechanisms, test higher doses safely, and evaluate long term effects before clinical trials in humans.
Background: Chronic Obstructive pulmonary disease (COPD) is an increasing cause of morbidity and mortality world-wide. MMP 9 is an acute phase reactant secreted by the liver in response to infection, inflammation or tissue damage. Methods: This case-control study was conducted on 35 healthy controls and 40 COPD patients at a tertiary care hospital in north India. MMP 9 levels were measured in serum by ELISA Kit. Results: The present study showed that mean MMP 9 levels in serum was significantly higher in COPD group as compared to control group (p<0.0001) and the levels increased with the increasing severity of the disease. Conclusion: Our study confirms that MMP 9 levels were significantly higher in COPD patients as compared to controls and their levels increased with the increasing severity of the disease. Measuring MMP 9 levels in combination with other biochemical markers can be helpful in monitoring disease outcome and management of the disease. Key-words- COPD, MMP 9, Inflammation, Matrix metalloproteinases (MMPs)
Cardiac Inflammation and Repair Following Myocardial InfarctionInsideScientific
Join Dr. Merry Lindsey as she discusses her research involving the physiology of recovery from cardiac events.
Age plays a pivotal role in the deterioration of cardiovascular functionality, resulting in an increased risk of cardiovascular disease in older adults. The prevalence of cardiovascular disease has also been shown to increase with age, in both men and women, including the prevalence of atherosclerosis, stroke and, myocardial infarction.
Following myocardial infarction (MI), the left ventricle (LV) undergoes a series of cardiac wound healing responses that involve both the stimulation of robust inflammation to clear necrotic myocytes and tissue debris and the induction of extracellular matrix (ECM) protein synthesis to generate an infarct scar. Collectively, this process in known as LV remodeling. Matrix metalloproteinase-9 (MMP-9) is a key regulator of LV remodeling post-MI, through direct effects on ECM turnover as well as indirect effects on the regulation of the major cell types that coordinate cardiac wound healing- namely the infiltrating leukocytes and the cardiac fibroblasts. We will discuss recent research that has expanded our understanding of MI LV remodeling, including recent proteomic advances focused on the ECM compartment to provide novel functional and translational insights. In summary, this webinar will provide an overview of how cardiac ECM research has evolved over the last decade and will provide insight into future directions that will drive further understanding of MMP directed cardiac ECM turnover after MI.
1. Octaplas is plasma derived from pooling donations which undergoes pathogen inactivation, filtration, and pooling. This decreases the risk of transfusion complications compared to single donor fresh frozen plasma.
2. Studies show Octaplas causes less damage to the endothelial glycocalyx and reduces bleeding and transfusion requirements compared to fresh frozen plasma in emergency surgeries.
3. Octaplas provides more consistent coagulation factor levels than single donor plasma and its use may decrease the need for additional coagulation factor concentrates like fibrinogen and prothrombin complex concentrate.
This document summarizes a study that evaluated inflammatory markers in different COPD subgroups compared to smokers and healthy controls. The study found that natural killer cells and CD56-expressing T cells were increased in COPD subjects regardless of rapid or slow lung function decline. These markers did not support the hypothesis that cytotoxic T cells can predict the rate of lung function decline in COPD. However, the increase in NKG2D in rapid decliners indicates these cells may play a role in lung function decline, warranting further research. A second study evaluated relationships between systemic acute-phase reactant biomarkers in COPD and found a pattern of relationships between these markers that differs from healthy smokers and with severity of functional impairment.
This document summarizes research on how shear stress regulates SIRT1 and its role in vascular homeostasis. The key points are:
1) Applying laminar flow to endothelial cells in vitro increased SIRT1 levels and activity as well as mitochondrial biogenesis. Higher SIRT1 levels were observed under physiologically relevant pulsatile flow compared to pathophysiological oscillatory flow.
2) Laminar flow activated both AMPK and SIRT1 independently in endothelial cells. Knockdown and genetic ablation studies showed they do not regulate each other.
3) Laminar flow increased the association between SIRT1 and eNOS and decreased eNOS acetylation. AMPK
1) The document examines ATF3 expression in cardiomyocytes in response to phenylephrine (PE) treatment.
2) Immunofluorescent staining of cardiomyocytes showed increased ATF3 nuclear staining after 0.5 hours of PE treatment, which decreased after 2 hours.
3) Quantitative PCR analysis found c-Jun mRNA was induced after 2 hours of PE, but ATF3, Egr1, and Fos mRNA levels remained unchanged.
My phD work Title "INVESTIGATION INTO THE MECHANISM OF ACTION OF CARDIOVASCUL...Hitesh Soni
Project Guide: Professor Anita A. Mehta ; The Best Project Guide I have ever seen.
Special Thanks to Dr. Mukul R Jain (Senior VP, Zydus Research Center) for continuous support. Thanks to Dr. Ajay Sharma (Associate Professor, Mason Eye Institute, USA) for concept building and giving training for Langendorrf's isolated heart experiments.
Inhaled nitric oxide can cause selective pulmonary vasodilation but clinical trials in adults with acute lung injury/ARDS found only short-lived physiological benefits with no reduction in mortality. While inhaled nitric oxide may help diagnose pulmonary hypertension, as a routine therapy for acute lung injury/ARDS in adults, the evidence does not support its effectiveness. Future studies of dosing strategies and adjunctive therapies are needed to determine if inhaled nitric oxide could improve outcomes.
This document summarizes research examining how the HIV envelope protein gp120 induces apoptosis in lung microvascular endothelial cells. The key findings are:
1) Gp120 upregulates surface expression of the proinflammatory cytokine EMAP II and its receptor CXCR3 on endothelial cells in a rapid and robust manner.
2) This upregulation occurs via gp120 signaling through its CXCR4 receptor and activation of the p38 MAPK pathway.
3) Both EMAP II and CXCR3 are essential for gp120-induced endothelial cell apoptosis.
4) The findings suggest a novel mechanism by which HIV infection causes endothelial cell loss and damage, contributing to lung diseases like emphysema.
The Influence of Various Drags on Mortality of Mice and the Concentration of ...CrimsonPublishersAICS
The Influence of Various Drags on Mortality of Mice and the Concentration of Proinflammatory Cytokines in Blood at Sepsis Caused by E. Coli by Zabrodskii PF* in Advancements in Case Studies
Acute and chronic hyperammonemia modulate antioxidantrkkoiri
1) The study examines the effects of acute and chronic hyperammonemia on antioxidant enzyme levels and oxidative damage in the cerebral cortex and cerebellum of rats.
2) Acute hyperammonemia decreased antioxidant enzyme levels in both brain regions but increased lipid peroxidation only in the cerebellum.
3) Chronic hyperammonemia increased antioxidant enzyme levels in the cerebral cortex but decreased levels in the cerebellum, suggesting the cerebellum is more susceptible to oxidative stress during chronic hyperammonemia.
This study investigated the effects of antioxidant supplementation on oxidative stress in patients with intermittent claudication. 16 patients performed a standard walking test before and after taking daily vitamins E and C for 4 weeks. Biomarkers were measured before, during, and after the tests. The study found that oxidative stress, as measured by the ortho-APOH biomarker, significantly increased during reperfusion after the initial walking test but did not increase after 4 weeks of antioxidant supplementation. This suggests that antioxidants reduce oxidative stress in claudicants caused by ischemia-reperfusion from walking.
This study investigated the effects of reducing indoor air particle levels through air filtration on microvascular function and biomarkers in elderly adults. The researchers conducted a randomized crossover study where 21 elderly couples were exposed to either filtered or nonfiltered indoor air in their homes for 48 hours. Microvascular function was assessed by measuring digital artery tone after induced ischemia and was significantly improved by 8.1% after indoor air filtration, suggesting short-term particle exposure reduction can benefit cardiovascular health in the elderly. Secondary biomarkers were not significantly changed after correction for multiple comparisons. The study provides evidence that indoor air particles negatively impact endothelial function and reducing exposure may help reduce cardiovascular disease risk.
This document discusses recent research findings about C-Reactive Protein (CRP) and its relationship to atherosclerosis and cardiovascular disease. Specifically, it summarizes studies that have found CRP has a direct vasodilatory effect on blood vessels through activation of potassium channels on vascular smooth muscle cells. This represents an important finding as it suggests CRP is actively involved in chronic inflammation associated with atherosclerosis rather than just being a marker of inflammation. The conclusion is that CRP likely plays a key role in the pathological processes of cardiovascular disease.
This document discusses recent research findings about C-Reactive Protein (CRP) and its relationship to atherosclerosis and cardiovascular disease. Specifically, it summarizes studies that have found CRP has a direct vasodilatory effect on blood vessels through activation of potassium channels on vascular smooth muscle cells. This represents an important finding as it suggests CRP is actively involved in chronic inflammation associated with atherosclerosis rather than just being a marker of inflammation. The conclusion is that CRP likely plays a key role in the pathological processes of cardiovascular disease.
TAEM10: How To Help Copd Patients Feel Better Andtaem
1. COPD is characterized by airflow limitation that is usually progressive and associated with an abnormal inflammatory response in the lungs caused by noxious particles or gases like cigarette smoke.
2. As COPD progresses, the total thickness of the airway walls increases, which correlates with decreased lung function. Inflammation is also present even in early stages of COPD.
3. The TORCH study found that treatment with the combination of salmeterol and fluticasone reduced the risk of death from COPD by 17.5% over three years compared to placebo, as well as reducing exacerbations.
Nitric oxide (NO) is an important signalling molecule in the body that regulates many physiological processes. It is synthesized by nitric oxide synthase enzymes from L-arginine and oxygen. NO signals through activation of soluble guanylate cyclase and production of cyclic GMP. It has diverse effects in the cardiovascular, immune, and nervous systems and is involved in processes like vasodilation, neurotransmission, and inflammation. Both insufficient and excessive NO production can contribute to disease.
It is well established that muscle contraction during exercise lead to elevated levels of reactive oxygen species in skeletal muscle ,hence the growing interest of oxidative stress in exercise
Light smoking and its affect on WBC, PFT and HbDr Sharath Rao
This study compared pulmonary function, white blood cell counts, and hemoglobin levels between asymptomatic light smokers and non-smokers. It found that light smokers had significantly higher total white blood cell counts than non-smokers, driven by higher absolute neutrophil, monocyte, and eosinophil counts. Light smokers also had significantly lower FEV1/FVC and FEF 75 measurements compared to non-smokers, indicating effects on small airway function. However, there were no significant differences found in hemoglobin levels, blood pressure, or other cardiovascular parameters between the two groups.
Los primeros dispositivos de cálculo incluyen el ábaco chino de hace más de 3,000 años y la máquina sumadora mecánica de Blaise Pascal en 1642. La máquina analítica de Charles Babbage en 1833 podía realizar cálculos automáticamente con placas perforadas. En 1944 se creó la primera computadora electrónica en la Universidad de Harvard, mientras que la primera computadora totalmente electrónica fue creada en la Universidad de Pensilvania en 1947. Las computadoras han evolucionado desde máquinas grandes y caras
AFEX Chile combate el lavado de dinero. Tratados anti lavado de dinero.valoreschile
El documento describe los principales tratados y organizaciones internacionales involucradas en la lucha contra el lavado de dinero, como la Convención de Viena de 1988, la Convención de Palermo de 2000 y el Grupo de Acción Financiera. También explica cómo la empresa Afex ha adoptado políticas y procedimientos internos para cumplir con estas normativas, detectar operaciones sospechosas y reportarlas a las autoridades competentes.
O documento apresenta um estudo sobre sementes com crianças da educação infantil. O objetivo é conhecer a diversidade de formas de vegetação e desenvolver habilidades de observação e classificação. Exemplos de sementes como feijão, arroz e milho são apresentados, assim como suas características de tamanho, cor e formato.
Background: Chronic Obstructive pulmonary disease (COPD) is an increasing cause of morbidity and mortality world-wide. MMP 9 is an acute phase reactant secreted by the liver in response to infection, inflammation or tissue damage. Methods: This case-control study was conducted on 35 healthy controls and 40 COPD patients at a tertiary care hospital in north India. MMP 9 levels were measured in serum by ELISA Kit. Results: The present study showed that mean MMP 9 levels in serum was significantly higher in COPD group as compared to control group (p<0.0001) and the levels increased with the increasing severity of the disease. Conclusion: Our study confirms that MMP 9 levels were significantly higher in COPD patients as compared to controls and their levels increased with the increasing severity of the disease. Measuring MMP 9 levels in combination with other biochemical markers can be helpful in monitoring disease outcome and management of the disease. Key-words- COPD, MMP 9, Inflammation, Matrix metalloproteinases (MMPs)
Cardiac Inflammation and Repair Following Myocardial InfarctionInsideScientific
Join Dr. Merry Lindsey as she discusses her research involving the physiology of recovery from cardiac events.
Age plays a pivotal role in the deterioration of cardiovascular functionality, resulting in an increased risk of cardiovascular disease in older adults. The prevalence of cardiovascular disease has also been shown to increase with age, in both men and women, including the prevalence of atherosclerosis, stroke and, myocardial infarction.
Following myocardial infarction (MI), the left ventricle (LV) undergoes a series of cardiac wound healing responses that involve both the stimulation of robust inflammation to clear necrotic myocytes and tissue debris and the induction of extracellular matrix (ECM) protein synthesis to generate an infarct scar. Collectively, this process in known as LV remodeling. Matrix metalloproteinase-9 (MMP-9) is a key regulator of LV remodeling post-MI, through direct effects on ECM turnover as well as indirect effects on the regulation of the major cell types that coordinate cardiac wound healing- namely the infiltrating leukocytes and the cardiac fibroblasts. We will discuss recent research that has expanded our understanding of MI LV remodeling, including recent proteomic advances focused on the ECM compartment to provide novel functional and translational insights. In summary, this webinar will provide an overview of how cardiac ECM research has evolved over the last decade and will provide insight into future directions that will drive further understanding of MMP directed cardiac ECM turnover after MI.
1. Octaplas is plasma derived from pooling donations which undergoes pathogen inactivation, filtration, and pooling. This decreases the risk of transfusion complications compared to single donor fresh frozen plasma.
2. Studies show Octaplas causes less damage to the endothelial glycocalyx and reduces bleeding and transfusion requirements compared to fresh frozen plasma in emergency surgeries.
3. Octaplas provides more consistent coagulation factor levels than single donor plasma and its use may decrease the need for additional coagulation factor concentrates like fibrinogen and prothrombin complex concentrate.
This document summarizes a study that evaluated inflammatory markers in different COPD subgroups compared to smokers and healthy controls. The study found that natural killer cells and CD56-expressing T cells were increased in COPD subjects regardless of rapid or slow lung function decline. These markers did not support the hypothesis that cytotoxic T cells can predict the rate of lung function decline in COPD. However, the increase in NKG2D in rapid decliners indicates these cells may play a role in lung function decline, warranting further research. A second study evaluated relationships between systemic acute-phase reactant biomarkers in COPD and found a pattern of relationships between these markers that differs from healthy smokers and with severity of functional impairment.
This document summarizes research on how shear stress regulates SIRT1 and its role in vascular homeostasis. The key points are:
1) Applying laminar flow to endothelial cells in vitro increased SIRT1 levels and activity as well as mitochondrial biogenesis. Higher SIRT1 levels were observed under physiologically relevant pulsatile flow compared to pathophysiological oscillatory flow.
2) Laminar flow activated both AMPK and SIRT1 independently in endothelial cells. Knockdown and genetic ablation studies showed they do not regulate each other.
3) Laminar flow increased the association between SIRT1 and eNOS and decreased eNOS acetylation. AMPK
1) The document examines ATF3 expression in cardiomyocytes in response to phenylephrine (PE) treatment.
2) Immunofluorescent staining of cardiomyocytes showed increased ATF3 nuclear staining after 0.5 hours of PE treatment, which decreased after 2 hours.
3) Quantitative PCR analysis found c-Jun mRNA was induced after 2 hours of PE, but ATF3, Egr1, and Fos mRNA levels remained unchanged.
My phD work Title "INVESTIGATION INTO THE MECHANISM OF ACTION OF CARDIOVASCUL...Hitesh Soni
Project Guide: Professor Anita A. Mehta ; The Best Project Guide I have ever seen.
Special Thanks to Dr. Mukul R Jain (Senior VP, Zydus Research Center) for continuous support. Thanks to Dr. Ajay Sharma (Associate Professor, Mason Eye Institute, USA) for concept building and giving training for Langendorrf's isolated heart experiments.
Inhaled nitric oxide can cause selective pulmonary vasodilation but clinical trials in adults with acute lung injury/ARDS found only short-lived physiological benefits with no reduction in mortality. While inhaled nitric oxide may help diagnose pulmonary hypertension, as a routine therapy for acute lung injury/ARDS in adults, the evidence does not support its effectiveness. Future studies of dosing strategies and adjunctive therapies are needed to determine if inhaled nitric oxide could improve outcomes.
This document summarizes research examining how the HIV envelope protein gp120 induces apoptosis in lung microvascular endothelial cells. The key findings are:
1) Gp120 upregulates surface expression of the proinflammatory cytokine EMAP II and its receptor CXCR3 on endothelial cells in a rapid and robust manner.
2) This upregulation occurs via gp120 signaling through its CXCR4 receptor and activation of the p38 MAPK pathway.
3) Both EMAP II and CXCR3 are essential for gp120-induced endothelial cell apoptosis.
4) The findings suggest a novel mechanism by which HIV infection causes endothelial cell loss and damage, contributing to lung diseases like emphysema.
The Influence of Various Drags on Mortality of Mice and the Concentration of ...CrimsonPublishersAICS
The Influence of Various Drags on Mortality of Mice and the Concentration of Proinflammatory Cytokines in Blood at Sepsis Caused by E. Coli by Zabrodskii PF* in Advancements in Case Studies
Acute and chronic hyperammonemia modulate antioxidantrkkoiri
1) The study examines the effects of acute and chronic hyperammonemia on antioxidant enzyme levels and oxidative damage in the cerebral cortex and cerebellum of rats.
2) Acute hyperammonemia decreased antioxidant enzyme levels in both brain regions but increased lipid peroxidation only in the cerebellum.
3) Chronic hyperammonemia increased antioxidant enzyme levels in the cerebral cortex but decreased levels in the cerebellum, suggesting the cerebellum is more susceptible to oxidative stress during chronic hyperammonemia.
This study investigated the effects of antioxidant supplementation on oxidative stress in patients with intermittent claudication. 16 patients performed a standard walking test before and after taking daily vitamins E and C for 4 weeks. Biomarkers were measured before, during, and after the tests. The study found that oxidative stress, as measured by the ortho-APOH biomarker, significantly increased during reperfusion after the initial walking test but did not increase after 4 weeks of antioxidant supplementation. This suggests that antioxidants reduce oxidative stress in claudicants caused by ischemia-reperfusion from walking.
This study investigated the effects of reducing indoor air particle levels through air filtration on microvascular function and biomarkers in elderly adults. The researchers conducted a randomized crossover study where 21 elderly couples were exposed to either filtered or nonfiltered indoor air in their homes for 48 hours. Microvascular function was assessed by measuring digital artery tone after induced ischemia and was significantly improved by 8.1% after indoor air filtration, suggesting short-term particle exposure reduction can benefit cardiovascular health in the elderly. Secondary biomarkers were not significantly changed after correction for multiple comparisons. The study provides evidence that indoor air particles negatively impact endothelial function and reducing exposure may help reduce cardiovascular disease risk.
This document discusses recent research findings about C-Reactive Protein (CRP) and its relationship to atherosclerosis and cardiovascular disease. Specifically, it summarizes studies that have found CRP has a direct vasodilatory effect on blood vessels through activation of potassium channels on vascular smooth muscle cells. This represents an important finding as it suggests CRP is actively involved in chronic inflammation associated with atherosclerosis rather than just being a marker of inflammation. The conclusion is that CRP likely plays a key role in the pathological processes of cardiovascular disease.
This document discusses recent research findings about C-Reactive Protein (CRP) and its relationship to atherosclerosis and cardiovascular disease. Specifically, it summarizes studies that have found CRP has a direct vasodilatory effect on blood vessels through activation of potassium channels on vascular smooth muscle cells. This represents an important finding as it suggests CRP is actively involved in chronic inflammation associated with atherosclerosis rather than just being a marker of inflammation. The conclusion is that CRP likely plays a key role in the pathological processes of cardiovascular disease.
TAEM10: How To Help Copd Patients Feel Better Andtaem
1. COPD is characterized by airflow limitation that is usually progressive and associated with an abnormal inflammatory response in the lungs caused by noxious particles or gases like cigarette smoke.
2. As COPD progresses, the total thickness of the airway walls increases, which correlates with decreased lung function. Inflammation is also present even in early stages of COPD.
3. The TORCH study found that treatment with the combination of salmeterol and fluticasone reduced the risk of death from COPD by 17.5% over three years compared to placebo, as well as reducing exacerbations.
Nitric oxide (NO) is an important signalling molecule in the body that regulates many physiological processes. It is synthesized by nitric oxide synthase enzymes from L-arginine and oxygen. NO signals through activation of soluble guanylate cyclase and production of cyclic GMP. It has diverse effects in the cardiovascular, immune, and nervous systems and is involved in processes like vasodilation, neurotransmission, and inflammation. Both insufficient and excessive NO production can contribute to disease.
It is well established that muscle contraction during exercise lead to elevated levels of reactive oxygen species in skeletal muscle ,hence the growing interest of oxidative stress in exercise
Light smoking and its affect on WBC, PFT and HbDr Sharath Rao
This study compared pulmonary function, white blood cell counts, and hemoglobin levels between asymptomatic light smokers and non-smokers. It found that light smokers had significantly higher total white blood cell counts than non-smokers, driven by higher absolute neutrophil, monocyte, and eosinophil counts. Light smokers also had significantly lower FEV1/FVC and FEF 75 measurements compared to non-smokers, indicating effects on small airway function. However, there were no significant differences found in hemoglobin levels, blood pressure, or other cardiovascular parameters between the two groups.
Los primeros dispositivos de cálculo incluyen el ábaco chino de hace más de 3,000 años y la máquina sumadora mecánica de Blaise Pascal en 1642. La máquina analítica de Charles Babbage en 1833 podía realizar cálculos automáticamente con placas perforadas. En 1944 se creó la primera computadora electrónica en la Universidad de Harvard, mientras que la primera computadora totalmente electrónica fue creada en la Universidad de Pensilvania en 1947. Las computadoras han evolucionado desde máquinas grandes y caras
AFEX Chile combate el lavado de dinero. Tratados anti lavado de dinero.valoreschile
El documento describe los principales tratados y organizaciones internacionales involucradas en la lucha contra el lavado de dinero, como la Convención de Viena de 1988, la Convención de Palermo de 2000 y el Grupo de Acción Financiera. También explica cómo la empresa Afex ha adoptado políticas y procedimientos internos para cumplir con estas normativas, detectar operaciones sospechosas y reportarlas a las autoridades competentes.
O documento apresenta um estudo sobre sementes com crianças da educação infantil. O objetivo é conhecer a diversidade de formas de vegetação e desenvolver habilidades de observação e classificação. Exemplos de sementes como feijão, arroz e milho são apresentados, assim como suas características de tamanho, cor e formato.
Este documento presenta información sobre las guías de estudio para la unidad de competencia sobre el pensamiento espacial y sistemas geométricos en el grado décimo de matemáticas. Incluye objetivos sobre definir gráficas de conicas como la circunferencia, parábola, elipse e hipérbola y resolver problemas aplicando sus ecuaciones. También presenta contenidos sobre distancia entre puntos, pendiente de rectas, ecuaciones de rectas y circunferencias, y elementos de la parábola.
Este documento presenta la Declaración y el Programa de Acción del Primer Congreso Mundial contra la Explotación Sexual Comercial de los Niños celebrado en Estocolmo, Suecia en 1996. Reafirma el compromiso de proteger los derechos de los niños contra la explotación sexual comercial y hace un llamamiento a los gobiernos y organizaciones para que tomen medidas urgentes para prevenir la explotación, proteger a las víctimas y castigar a los responsables.
Topic of the month.... Endothelial nitric oxide synthase (eNOS) And Stroke: P...Professor Yasser Metwally
Topic of the month.... Endothelial nitric oxide synthase (eNOS) And Stroke: Prevention, Treatment And Recovery
http://yassermetwally.com
http://yassermetwally.net
El documento habla sobre el rock clásico, definido como los grupos de rock que surgieron entre las décadas de 1950 y 1970. Explica que no constituye un estilo en sí mismo, sino que agrupa bandas de diversos géneros como el rock and roll. Describe algunas características de Queen como banda emblemática del rock clásico y resume brevemente la historia del desarrollo del rock y algunos hitos importantes de los años 1960 como el surgimiento de The Beatles y la invasión británica.
This document summarizes a study investigating the role of autophagy in lung inflammation and corticosteroid-resistant neutrophilic asthma. The study found that deletion of the autophagy gene Atg5 specifically in CD11c+ cells caused spontaneous airway hyperreactivity and severe neutrophilic lung inflammation in mice. Mice lacking Atg5 had higher IL-17A and IL-1β levels in the lungs and increased IL-17A-producing T cells, indicating autophagy impairment induces neutrophilic inflammation through IL-17A secretion. The study also found that lack of autophagy in immune cells, but not lung epithelial cells, contributed to lung inflammation. This suggests
This study investigated the effects of inhaling residual oil fly ash (ROFA) particles on oxidative stress and hemodynamics in exercising rats. The key findings were:
1. Rats that inhaled ROFA before exercise showed increased lipid peroxidation (a marker of oxidative stress) in the lungs and heart compared to those that inhaled saline.
2. However, ROFA inhalation did not significantly alter heart rate, blood pressure, or other hemodynamic responses during exercise compared to saline.
3. While ROFA promoted oxidative stress in the lungs and heart, it did not produce significant changes in cardiovascular function during swimming exercise in rats.
Use of Capnograph in Breathlessness Patientsnhliza
This is a research topic carried out in the Emergency Department and the abstract was presented at the International Conference In Emergency Medicine in SanFrancisco April 2008
This document summarizes a study that investigated sex-based differences in bronchiolar epithelial injury and repair following naphthalene inhalation in rats. The key points are:
1) Male and female rats were exposed to naphthalene vapor and sacrificed at various time points to assess injury and regeneration of bronchiolar epithelium.
2) Naphthalene exposure induced bronchiolar cell exfoliation peaking at 24 hours in both sexes. Male epithelium fully regenerated by 14 days, while females still had some exfoliated cells at 14 days that disappeared by 21 days.
3) Injury occurred earlier and involved more cells in females, while regeneration occurred earlier in males
The document summarizes an experiment that exposed mice susceptible to atherosclerosis to different components of ultrafine particles (UFP) to evaluate their effects on oxidative stress. Mice were exposed to either the semi-volatile or non-volatile fraction of UFP for 8 weeks. Biomarkers of oxidative stress (glutathione, malondialdehyde, protein carbonyl) were measured in mice serum to determine if one fraction induced more stress. Results suggested the semi-volatile fraction containing polycyclic aromatic hydrocarbons caused higher lipid peroxidation, supporting the hypothesis that these components influence oxidative stress more than non-volatile ones.
Reexpansion pulmonary edema (RPE) is a rare but potentially lethal condition that can occur after the rapid expansion of a collapsed lung. The pathophysiology is multifactorial but is believed to involve decreased pulmonary surfactant levels and a pro-inflammatory response due to the lung collapse and reexpansion. Early diagnosis is important as prognosis depends on prompt treatment, but prevention through slow, controlled reexpansion is the best strategy due to the high mortality rates associated with RPE.
This study evaluated the protective effects of amlodipine, lisinopril, and allopurinol against acetaminophen-induced hepatotoxicity in rats. Rats pre-treated with these drugs showed significantly lower liver damage markers and oxidative stress compared to rats that only received acetaminophen. Histological analysis also supported the biochemical results. This suggests that calcium channels, angiotensin converting enzyme, and xanthine oxidase play roles in the mechanism of acetaminophen hepatotoxicity. Amlodipine, lisinopril, and allopurinol may protect the liver by modulating these pathways.
This study examined the effects of prolonged simvastatin (SIM) treatment on ischemia-reperfusion (I/R) induced acute kidney injury in rats. Rats were divided into four groups: sham, ischemia, I/R, and I/R+SIM treated. The I/R group showed intense inflammation, necrosis, and apoptosis in kidney tissue. The I/R+SIM group showed reduced inflammation and tissue damage. Biochemical analysis found increased oxidative stress and inflammation markers in the ischemia and I/R groups compared to control, but levels in the I/R+SIM group were similar to control. Histological analysis also showed more damage in ischemia and I/R groups versus control, while the I/R+
This document summarizes a project investigating whether mitochondrial DNA-encoded oxidative phosphorylation (OXPHOS) transcripts are dysregulated in the blood of patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD) compared to controls. The student researcher designed primers, conducted quantitative real-time PCR on blood samples from controls and patients, and found several mtDNA-encoded OXPHOS transcripts were significantly more abundant in MCI and AD patients. This suggests peripheral changes in mitochondrial gene expression occur early in AD and could provide biomarkers for early diagnosis and monitoring disease progression and treatment responses.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
[Interdisciplinary Toxicology] Evaluation of miR-9 and miR-143 expression in ...mostafa khafaei
The document discusses a study that evaluated the expression levels of miR-9 and miR-143 in urine samples from 32 sulfur mustard exposed patients and 32 healthy subjects. The study found that the expression levels of both miR-9 and miR-143 were significantly decreased in the sulfur mustard exposed patients compared to the healthy subjects, with p-values of 0.0480 and 0.0272, respectively. This suggests an imbalance in several pathways involved in the pathogenic effects of sulfur mustard exposure, such as NF-κB signaling, TGF-β signaling, WNT pathway, inflammation, DNA repair, and apoptosis. The decreases in miR-9 and miR-143 expression may play an important role in the pathogenicity of patients exposed to
Background
The normal values of α-amylase in humans is unknown.
Objective
To determine the normal values of α-amylase activity in humans.
Material and methods
From October 2009 to June 2011 we studied 107 patients referenced to thoracic service to be submitted to bronchoscopy.
The patients were positioned in supine position, performed local antisepsis and anesthetized with 2% lidocaine. Thereafter we introduced a needle into catheter by puncturing the cricothyroid membrane using a 14G cateter. Finally we introduced ten milliliters of saline and immediately aspirated with the maximum power of the vacuum system. The samples were sent for α- amylase activity determination by CNPG method.
Results
The activity of α-amylase of tracheal aspirate ranged from 24 to 10.000 IU/l), and a mean 1914IU/L. The levels had no statistical differences according age, sex, race, smoking history and the lung diseases.
Conclusion
We could define the probably physiologic levels of α-amylase activity in humans beings.
The document defines acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) according to criteria from a 1994 consensus conference. It discusses the epidemiology and clinical disorders associated with ALI/ARDS development. Ventilator-based strategies for management include using low tidal volumes (6 ml/kg) and positive end-expiratory pressure (PEEP) of 13-16 cm H2O to reduce ventilator-induced lung injury from overdistension and repetitive opening/closing of alveoli. Recruitment maneuvers involving brief increases in pressure have been used to improve oxygenation by opening collapsed lung regions.
The document summarizes research into a novel drug target for the treatment of Alzheimer's disease. It discusses how endoplasmic reticulum (ER) stress is implicated in the disease and how activating the calcium pump SERCA could help reduce ER stress. It then describes a drug candidate called CDN1163 that was discovered to directly activate SERCA. Studies in mouse models found CDN1163 enhanced calcium uptake, showed promising pharmacokinetics, and improved memory in behavioral tests, suggesting it may help treat Alzheimer's by reducing ER stress.
1) The study investigated the effects of gasdermin D on pyroptosis in a mouse model of sepsis-induced acute kidney injury.
2) The results showed that gasdermin D expression was increased in mice with sepsis-induced acute kidney injury and promoted inflammation and pyroptosis in kidney cells.
3) Downregulating gasdermin D decreased inflammation and pyroptosis, and the NLRP3 inflammasome was identified as an important target of gasdermin D in mediating inflammation during sepsis-induced acute kidney injury.
Background: Body of literature are becoming pronounced that pathological condition in one organ of the body might have an effect on other distal organs owing to the fact, that the entire body metabolism is orchestrated centrally.
Pathological events occurring in an organ are likely to be extended to other organs. Pretreatment that minimize these events are presumed to be beneficial to the extended organs.
Methods: Following 30 min of ischemia and 48 h of reperfusion in the kidney, rats under anesthesia were sacrificed and blood sample collected through cardiac puncture. Serum level of troponin I, and activities of total creatine kinase (CK), mass creatine kinase (CK-MB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and gamma –glutamyl transferase (GGT) were estimated spectrophotometrically.
Results: Serum troponin I increased to 0.031 ± 0.001 ng/ml in the ischemic group, and following pretreatment with Lmm (600mg/kg), serum level of troponin I decreased significantly to 0.021 ± 0.001 ng/ml (P<.05).><.05),><.05)><.05).
SHER-E-KASHMIRUNIVERSITY OF AGRICULTURAL SCIENCES ANDTECHNOLOGY, KASHMIR.pptxcdrecordsection
This case study evaluated the hepatoprotective effects of formulations containing extracts of Berberis aristata, Solanum nigrum, and Aloe vera (Formulation A), and a decoction of Phyllanthus fraternus (Formulation B), on patients receiving antitubercular treatment. Patients taking the formulations showed no significant increases in liver enzymes, while the control group showed significant increases. The formulations helped prevent antitubercular treatment-induced hepatotoxicity and allowed patients to complete their treatment courses.
CELLULAR ADAPTATIONS IN THE DIAPHRAGM IN CHRONIC OBSTRUCTIVETeresa Seguro
The document summarizes a study comparing the cellular adaptations in the diaphragm muscle between patients with severe chronic obstructive pulmonary disease (COPD) and healthy control subjects. Biopsy samples of the diaphragm were obtained from 6 COPD patients and 10 controls. Analyses found that the diaphragm of COPD patients had a higher percentage of slow-twitch muscle fibers and slow isoforms of myofibrillar proteins compared to controls. Specifically, the diaphragm of COPD patients had a higher percentage of slow myosin heavy chain I and lower percentages of fast myosin heavy chains IIa and IIb. A similar trend was seen when comparing fiber types using immunohistochemical techniques. These cellular changes
Protective role of co q10 or l carnitine on the integrity of the myocardium i...Prof. Hesham N. Mustafa
Doxorubicin (DOX) is a chemotherapeutic agent used for treatment of different cancers and its clinical usage is hindered by the oxidative injury-related cardiotoxicity. This work aims to declare if the harmful effects of DOX on heart can be alleviated with the use of Coenzyme Q10 (CoQ10) or L-carnitine. The study was performed on seventy two female Wistar albino rats divided into six groups, 12 animals each: Control group; DOX group (10mg/kg); CoQ10 group (200mg/kg); L-carnitine group (100mg/kg); DOX+CoQ10 group; DOX+L-carnitine group. CoQ10 and L-carnitine treatment orally started 5days before a single dose of 10mg/kg DOX that injected intraperitoneally (IP) then the treatment continued for 10days. At the end of the study, serum biochemical parameters of cardiac damage, oxidative stress indices, and histopathological changes were investigated. CoQ10 or L-carnitine showed a noticeable effects in improving cardiac functions evidenced reducing serum enzymes as serum interleukin-1 beta (IL-1 β), tumor necrosis factor alpha (TNF-α), leptin, lactate dehydrogenase (LDH), Cardiotrophin-1, Troponin-I and Troponin-T. Also, alleviate oxidative stress, decrease of cardiac Malondialdehyde (MDA), Nitric oxide (NO) and restoring cardiac reduced glutathione levels to normal levels. Both corrected the cardiac alterations histologically and ultrastructurally. With a visible improvements in α-SMA, vimentin and eNOS immunohistochemical markers. CoQ10 or L-carnitine supplementation improves the functional and structural integrity of the myocardium.
Keywords: Cardiotoxicity; CoQ10 and L-carnitine; Dox; Vimentin; eNOS.
Similar to Mechanisms of nitric oxide synthase (20)
TEST BANK For An Introduction to Brain and Behavior, 7th Edition by Bryan Kol...rightmanforbloodline
TEST BANK For An Introduction to Brain and Behavior, 7th Edition by Bryan Kolb, Ian Q. Whishaw, Verified Chapters 1 - 16, Complete Newest Versio
TEST BANK For An Introduction to Brain and Behavior, 7th Edition by Bryan Kolb, Ian Q. Whishaw, Verified Chapters 1 - 16, Complete Newest Version
TEST BANK For An Introduction to Brain and Behavior, 7th Edition by Bryan Kolb, Ian Q. Whishaw, Verified Chapters 1 - 16, Complete Newest Version
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Kat...rightmanforbloodline
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
TEST BANK For Basic and Clinical Pharmacology, 14th Edition by Bertram G. Katzung, Verified Chapters 1 - 66, Complete Newest Version.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Integrating Ayurveda into Parkinson’s Management: A Holistic ApproachAyurveda ForAll
Explore the benefits of combining Ayurveda with conventional Parkinson's treatments. Learn how a holistic approach can manage symptoms, enhance well-being, and balance body energies. Discover the steps to safely integrate Ayurvedic practices into your Parkinson’s care plan, including expert guidance on diet, herbal remedies, and lifestyle modifications.
Adhd Medication Shortage Uk - trinexpharmacy.comreignlana06
The UK is currently facing a Adhd Medication Shortage Uk, which has left many patients and their families grappling with uncertainty and frustration. ADHD, or Attention Deficit Hyperactivity Disorder, is a chronic condition that requires consistent medication to manage effectively. This shortage has highlighted the critical role these medications play in the daily lives of those affected by ADHD. Contact : +1 (747) 209 – 3649 E-mail : sales@trinexpharmacy.com
Here is the updated list of Top Best Ayurvedic medicine for Gas and Indigestion and those are Gas-O-Go Syp for Dyspepsia | Lavizyme Syrup for Acidity | Yumzyme Hepatoprotective Capsules etc
- Video recording of this lecture in English language: https://youtu.be/kqbnxVAZs-0
- Video recording of this lecture in Arabic language: https://youtu.be/SINlygW1Mpc
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
Muktapishti is a traditional Ayurvedic preparation made from Shoditha Mukta (Purified Pearl), is believed to help regulate thyroid function and reduce symptoms of hyperthyroidism due to its cooling and balancing properties. Clinical evidence on its efficacy remains limited, necessitating further research to validate its therapeutic benefits.
Rasamanikya is a excellent preparation in the field of Rasashastra, it is used in various Kushtha Roga, Shwasa, Vicharchika, Bhagandara, Vatarakta, and Phiranga Roga. In this article Preparation& Comparative analytical profile for both Formulationon i.e Rasamanikya prepared by Kushmanda swarasa & Churnodhaka Shodita Haratala. The study aims to provide insights into the comparative efficacy and analytical aspects of these formulations for enhanced therapeutic outcomes.
Cell Therapy Expansion and Challenges in Autoimmune DiseaseHealth Advances
There is increasing confidence that cell therapies will soon play a role in the treatment of autoimmune disorders, but the extent of this impact remains to be seen. Early readouts on autologous CAR-Ts in lupus are encouraging, but manufacturing and cost limitations are likely to restrict access to highly refractory patients. Allogeneic CAR-Ts have the potential to broaden access to earlier lines of treatment due to their inherent cost benefits, however they will need to demonstrate comparable or improved efficacy to established modalities.
In addition to infrastructure and capacity constraints, CAR-Ts face a very different risk-benefit dynamic in autoimmune compared to oncology, highlighting the need for tolerable therapies with low adverse event risk. CAR-NK and Treg-based therapies are also being developed in certain autoimmune disorders and may demonstrate favorable safety profiles. Several novel non-cell therapies such as bispecific antibodies, nanobodies, and RNAi drugs, may also offer future alternative competitive solutions with variable value propositions.
Widespread adoption of cell therapies will not only require strong efficacy and safety data, but also adapted pricing and access strategies. At oncology-based price points, CAR-Ts are unlikely to achieve broad market access in autoimmune disorders, with eligible patient populations that are potentially orders of magnitude greater than the number of currently addressable cancer patients. Developers have made strides towards reducing cell therapy COGS while improving manufacturing efficiency, but payors will inevitably restrict access until more sustainable pricing is achieved.
Despite these headwinds, industry leaders and investors remain confident that cell therapies are poised to address significant unmet need in patients suffering from autoimmune disorders. However, the extent of this impact on the treatment landscape remains to be seen, as the industry rapidly approaches an inflection point.
2. pulmonary capillary integrity thus leading to significant morbidity and mortality (Martinez et
al., 2009). In ALI/ARDS, the integrity of the separation between the alveolus and the
pulmonary circulation is compromised either by endothelial and/or epithelial injury. This
damage leads to increased vascular permeability, alveolar flooding, and surfactant
abnormalities (due to damage of type II pneumocytes). ALI can occur in response to a number
of insults that either directly or indirectly induce lung injury. The most common indirect
pulmonary insult leading to ALI is the release of lipopolysaccharide (LPS; endotoxin) from
the outer cell wall of most gram-negative bacteria producing sepsis (Erickson et al., 2009).
Despite great advances in understanding the pathophysiology of ALI/ARDS, the available
therapies have not led to a significant reduction in mortality or an increased quality of life in
survivors. Thus, a greater understanding of the mechanisms by which the pathways leading to
ALI are disrupted could lead to the development of more effective therapies.
ADMA is an endogenously produced competitive inhibitor of NO synthases (Vallance et al.,
1992) and has been shown to be a cardiovascular risk factor for numerous diseases. ADMA is
constantly produced in the course of normal protein turnover in many tissues, including
vascular endothelial cells, and is derived from the hydrolysis of methylated proteins (Kakimoto
and Akazawa, 1970). ADMA is metabolized via hydrolytic degradation to citrulline and
dimethylamine by the enzyme dimethylarginine dimethylaminohydrolase (DDAH) (Kimoto
et al., 1995). Elevated ADMA levels have been shown to attenuate endothelium-dependent
vasodilation in humans (Boger, 2003a; Boger and Bode-Boger, 2000). In addition, inhibition
of DDAH results in vasoconstriction of vascular segments that can be reversed by L-arginine
(MacAllister et al., 1996). Earlier studies have shown that ADMA can disrupt NO signaling
and induce endothelial dysfunction (Boger, 2003a,b, 2004; Boger and Bode-Boger, 2000; Tran
et al., 2003). There is also increasing evidence that ADMA causes NOS uncoupling in
endothelial cells leading to increased superoxide generation (Sud et al., 2008; Antoniades et
al., 2009). Superoxide free radicals can react with NO to form peroxynitrite (ONOO–), which
is a potent reactive nitrogen species (RNS) that causes the irreversible nitration of tyrosine
residues within proteins that can in turn lead to cellular damage and cytotoxicity. Nitrotyrosine
(3-NT) is a major product formed by peroxynitrite mediated nitration of proteins (Szabo,
2003). Our previous studies have shown that ADMA uncouples eNOS leading to an increase
in superoxide production resulting in increased peroxynitrite generation and nitrotyrosine
protein levels in endothelial cells (Sud et al., 2008).
In a recent study, LPS was found to increase the levels of ADMA and decrease DDAH activity
in human endothelial cells. LPS also increased intracellular reactive oxygen species production
in these cells (Xin et al., 2007). Another study has shown that ADMA levels were elevated in
patients with septic shock (O'Dwyer et al., 2006). Peroxynitrite has been shown to play a role
in the pathogenesis of endotoxin-induced homodynamic instability and organ dysfunction
(Zingarelli et al., 1997). Previous studies in animal models of ALI have shown the elevated
levels of 3-NT levels in the pulmonary tissue and BAL fluid (Laffey et al., 2004; Chen et al.,
2003; Tsuji et al., 2000; Shang et al., 2008) while increases in 3-NT levels in ALI have
previously been shown to be iNOS-dependent (Tsuji et al., 2000; Chen et al., 2003; Razavi et
al., 2005). However, at present there have been no studies that evaluate the early effects on
ADMA levels and NOS signaling in the murine model of ALI induced by LPS. Thus, in this
study we utilized the LPS-induced mouse model of ALI to investigate whether alterations in
the ADMA/DDAH pathway may contribute to the pathogenesis of this disease. Furthermore,
we explored the role played by increased ADMA levels in increasing nitrosative stress and
subsequent nitration of proteins as a possible mechanism leading to LPS induced ALI. Finally,
we evaluated whether the scavenging of ONOO– could exert a protective effect on the lung
leak associated with ALI.
Sharma et al. Page 2
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3. 2. Materials and methods
2.1. In vivo experiments
2.1.1. LPS treatment—Adult male C57BL/6NHsd mice (7–8 weeks; Harlan Indianapolis,
IN) were used in all experiments. All animal care and experimental procedures were approved
by the Committee on Animal Use in Research and Education of the Medical College of Georgia
(Augusta, GA). Stock solutions of lipopolysaccharide (LPS), purified from Escherichia coli
(serotype 0111:B4) was prepared in 0.9% saline. Mice received vehicle (10% DMSO in saline)
or LPS (6.75×104 EU/gm body wt) intraperitoneally. Mice were then euthanized at 0, 2, 4, and
12 h after LPS injection and the lungs were flushed with 1 ml of ice-cold EDTA-PBS excised,
snap-frozen in liquid nitrogen, and stored at −80 °C until used.
2.1.2. Peroxynitrite scavenger treatments—Manganese (III) tetrakis (1-methyl-4-
pyridyl) porphyrin (MnTymPyp, A.G. Scientific, Inc. San Diego, CA), was prepared in distilled
water, 0 h control mice received an intraperitoneal injection (IP) of water. Uric acid was
dissolved in 25% glycerol and 75%, of 0.9% saline, 0 h control mice received an intraperitoneal
injection (IP) of 25% glycerol and 75% of 0.9% saline. In the experiments to determine lung
leak (Evans Blue), MnTymPyp (5 mg/kg body weight), uric acid (5 mg/kg body weight) or
corresponding vehicle was injected I.P. 30 min prior to LPS injections. Subsequent doses of
uric acid were injected 3 and 6 h post LPS injection (Hooper et al., 1998). After 12 h of LPS
exposure, animals were anesthetized and Evans Blue surgery was performed. To determine
total nitration levels, MnTymPyp, uric acid, or vehicle was injected I.P. 30 min prior to LPS
injections. A subsequent dose of uric acid was injected 3 h post LPS injection. Animals were
then euthanized, blood was collected by ventricular puncture and the lungs were flushed with
ice-cold phospho-buffered saline and EDTA. The lungs for total nitration were then excised,
snap frozen in liquid nitrogen and stored at −80 °C until used.
2.2. Lung tissue homogenates
Lung protein extracts were prepared by homogenizing mouse lung tissues in Triton lysis buffer
(50 mM Tris–HCL, pH 7.6, 0.5%Triton X-100, 20% glycerol) containing a protease inhibitor
cocktail (Sigma). Extracts were then clarified by centrifugation (15,000 g×10 min at 4 °C).
Supernatant fractions were then assayed for protein concentration using the Bradford reagent
(Bio-Rad, Richmond, CA).
2.3. Western blot analyses
Western blot analysis was performed as previously described (Sharma et al., 2008, 2007; Sud
et al., 2008)). Briefly, protein extracts (25–50 μg) were separated on 4–20% denaturing
polyacrylamide gels and transferred to Immunoblot-PVDF membranes (Biorad Lab, Hercules,
CA). The membranes were blocked with 5% nonfat dry milk in TBS containing 0.1% Tween.
After blocking, the membranes were incubated overnight at 4 °C with eNOS (1:1000, BD
Transduction), nNOS (1:1000, BD Transduction), iNOS (1:1000, Upstate), DDAH I (1:500,
Biosynthesis Inc., Louisville, TX) and DDAH II (Biosynthesis Inc., Louisville, TX), 3-
nitrotyrosine (3-NT) antibody (1:1000, Calbiochem, San Diego, CA), mouse β-actin (1:10,000,
Sigma), washed with TBS containing 0.1% Tween, and then incubated with a goat anti-mouse
IgG-horseradish peroxidase. After washing, the protein bands were visualized with
chemiluminescence (West Femto kit, Pierce) using a Kodak Digital Science Image Station.
All protein bands were densitometrically analyzed using Kodak Imaging software. To
normalize for protein loading, blots were re-probed with β-actin, the housekeeping protein.
Sharma et al. Page 3
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4. 2.4. Measurement of ADMA levels
ADMA levels were analyzed by high-performance liquid chromatography (HPLC) as we have
previously published (Sud et al., 2008). The crude fraction of cell lysate was isolated using a
solid phase extraction column and subsequently, ADMA was separated using pre-column
derivatization with ortho-phthaldialdehyde (OPA) reagent (4.5 mg/mL in borate buffer, pH
8.5, containing 3.3 μl/mL β-mercaptoethanol) prior to injection. HPLC was performed using
a Shimadzu UFLC system with a Nucleosil phenyl reverse phase column (4.6 × 250 mm;
Supelco, Bellefonte, PA), equipped with an RF-10AXL fluorescence detector (Shimadzu USA
Manufacturing Corporation). ADMA levels were quantified by fluorescence detection at 450
nm (emission) and 340 nm (excitation). Mobile phase A was composed of 95% potassium
phosphate (50 mM, pH 6.6), 5% methanol and mobile phase B was composed of 100%
methanol. ADMA was separated using a pre-gradient wash of 25% mobile phase B(flow rate
0.8 mL/min), followed by a linear increase in mobile phase B concentration from 20% to 25%
over 7 min followed by a constant flow at 25% for 10 min and another linear increase from
25% to 27% mobile phase B over 5 min followed by constant flow at 27% mobile phase B for
another 7 min. Retention time for ADMA was approximately 28 min. ADMA concentrations
were calculated using standards and an internal homoarginine standard. The detection limit of
the assay was 0.1 μmol/L.
2.5. Measurement of DDAH activity
DDAH activity in LPS treated mouse lungs was assessed directly by measuring the amount of
ADMA metabolized by this enzyme as previously described (Lin et al., 2002). DDAH activity
is defined as the amount of ADMA degraded per mg protein.
2.6. Measurement of BH4 levels
BH4 levels were determined using the differential iodine oxidation method as we have
previously described (Kumar et al., 2009; Wainwright et al., 2005). Lung tissue was
homogenized in an extraction buffer (50 mM pH 7.4 Tris-HCl, 1 mM EDTA, 1 mM DTT) and
divided into equal volumes between two centrifuge tubes containing either 1 M NaOH or 1 M
H3PO4. A solution of 1% I2 in 2% KI was added to each tube and samples were then incubated
in the dark at RT for 90 min. 1 M H3PO4 was then added to the tubes containing NaOH. Excess
I2 was removed from the samples by adding 2% ascorbic acid and samples were centrifuged
at 15,000 ×g for 10 min to remove the precipitated protein. Eac supernatant was then analyzed
for BH4 content by HPLC using a Spherisorb ODS-1 column (Waters, Franklin MA). BH4
levels were calculated by subtracting the area of the biopterin peak resulting from the oxidation
of BH2 in the base solution from the peak resulting from the oxidation of both BH2 and BH4
in the acidic solution. Levels were normalized for protein concentration by Bradford assay.
2.7. Assessment of lung capillary leakage
Mice were anesthetized 12 h after LPS administration, with ketamine (80 mg/kg) and xylazine-
HCl (8 mg/kg). Evans blue dye (EB) dissolved in saline was injected (100 mg/kg) through the
left jugular vein, using a 30-gauge needle inserted to PE-10 tubing. After 30 min, blood was
withdrawn via cardiac puncture and stored at 4 °C. The lungs were flushed with 1 ml of EDTA–
PBS (pH 7.4, 4 °C), excised, snap-frozen in liquid nitrogen, and stored at −80 °C. Frozen lungs
were homogenized in ice-cold PBS (1 ml/100 mg tissue), incubated with 2 volumes of
formamide (60 °C, 18 h), and centrifuged (5,000 ×g for 30 min), and supernatant absorbance
at 620 nm (A620) and 740 nm (A740) was recorded. Tissue EB content was calculated by
correcting the A620 optical density for the presence of heme pigments: A620 (corrected) =
A620 − (1.426 × A740 + 0.030) and by then comparing this value with a standard curve of EB
in formamide–PBS. Total EB leak was expressed as lungEB content divided by serumEB
content.
Sharma et al. Page 4
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5. 2.8. Superoxide quantitation in lung tissue
Superoxide levels in mouse lung tissue taken from 0, 2, 4, and 12 h post LPS treatments, were
estimated by electronic paramagnetic resonance (EPR) assay using the spin-trap compound 1-
hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine HCl (CMH) as we have
previously described (Lakshminrusimha et al., 2007). Briefly, 0.1 g of tissue was sectioned
from fresh-frozen lung tissue and immediately immersed, while still frozen, in 200 μl of EPR
Buffer (PBS supplemented with 5 μM diethydithiocarbamate [DETC, Sigma-Aldrich], and 25
μM desferrioxamine [Def MOS, Sigma-Aldrich]). All samples were then incubated for 30 min
on ice then homogenized for 30 s with a VWR PowerMAX AHS 200 tissue homogenizer.
Following incubation, samples were analyzed for protein content using Bradford analysis.
Sample volumes were then adjusted with EPR buffer and 25 mg/ml CMH-hydrochloride in
order to achieve equal protein content and a final CMH concentration of 5 mg/ml. Samples
were further incubated for 60 min on ice and centrifuged at 14,000×g for 15 min at room
temperature. 35 μl of supernatant from each sample was loaded into a 50 μl capillary tube and
analyzed with a MiniScope MS200 ESR (Magnettech, Berlin, Germany) at a microwave power
of 40 mW, modulation amplitude of 3000 mG, and modulation frequency of 100 kHz, with a
magnetic strength of 333.95–3339.94 mT. Resulting EPR spectra were analyzed using
ANALYSIS v.2.02 software (Magnettech), whereby the EPR maximum and minimum spectral
amplitudes for the CM superoxide spin-trap product waveform were quantified. Experimental
groups were normalized to fold vs. untreated control samples, then compared for differences
in O2
−. concentration using statistical analysis. The specificity for superoxide and the level of
NOS-derived superoxide were determined by incubating duplicate samples with either PEG-
SOD (100 U) or the NOS inhibitor NG-monomethyl L-arginine (L-NMMA; 100 μM)
respectively.
2.9. Measurement of NOx levels
In order to quantify bioavailable NO, NO and its metabolites were determined in mouse lung
tissue. In solution, NO reacts with molecular oxygen to form nitrite, and with oxyhemoglobin
and superoxide anion to form nitrate. Nitrite and nitrate are reduced using vanadium (III) and
hydrochloric acid at 90 °C. NO is purged from solution resulting in a peak of NO for subsequent
detection by chemiluminescence (NOA 280, Sievers Instruments Inc. Boulder CO), as we have
previously described (Black et al., 1999; McMullan et al., 2000). The sensitivity is 1 × 10−12
mol, with a concentration range of 1×10−9 to 1×10−3 mol of nitrate.
2.10. Human lung microvascular endothelial cell isolation and culture
Isolation and culture of human lung microvascular endothelial cell (HLMVEC) was performed
by a modification of the method of Hewett and Murray (Hewett and Murray, 1993) and Burg
et al (Burg et al., 2002). Normal human lung tissue was obtained from lobectomy specimens
resected due to lung disease. Briefly, isolation of HPMEC was performed as follows: subpleural
lung tissue was cut into small fragments with scissors. After removal of debris and erythrocytes
by filtering through a 40 μm nylon net, the tissue was treated with dispase (1 U/ml at 4 °C for
18 h). After filtration through a 100 μm nylon net, the tissue was treated in a volume of 15 ml
M199, 15% FBS, 1 mg dispase/ml at 37 °C for 1 h followed by a further flitration through a
100 μm nylon net. The cell clumps within the filtrate were repeatedly resuspended in M199
and filtered through a 40 μm net, followed by centrifugation for 10 min and resuspension in
M199 containing 20% serum. Undigested tissue was washed from the 100 μm net, collected
and digested again in 1 mg dispase/ml as above. The positive selection of HLMVEC was
achieved by interacting the cell suspension with magnetic beads (Tosyl activated Dynabeads:
Invitrogen) coated with Ulex europaeus I according to the method of Jackson et al (Jackson et
al., 1990). After purification, cells were cultured in M199, 20% FBS, 100U Heparin/ml, 150
μg ECGF/ml, 1 μg hydrocortisone/ml, 292mg L-glutamine/l, and 110 mg sodium pyruvate/l.
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6. EC identity was confirmed by uptake of 1,1_-dioctadecyl-1,3,3_,3_-tetramethyl-
indocarbocyanineacetylated low-density lipoprotein (Dil-Ac-LDL) and used between passages
1–3.
2.11. Measurement of transendothelial cell electrical impedance
Transendothelial impedance was measured using an electric cell impedance sensing (ECIS)
apparatus (Applied Biophysics, Troy, NY). Equal number of HLMVEC were seeded on L-
cysteine coated gold electrode arrays (8W10E) and allowed to grow to confluence then serum
starved for 4 h. Transendothelial impedance was monitored for 30 min to establish baseline.
The cells were treated or not with ADMA (5 μM) in the presence or absence of vascular
endothelial growth factor (VEGF,100 ng) and the effect on endothelial permeability measured
over 2 h.
2.12. Statistical analysis
Statistical analysis was performed using GraphPad Prism version 4.01 for Windows (GraphPad
Software, San Diego, CA). The mean±SEM were calculated for all samples and significance
was determined either by the unpaired t-test (for 2 groups) and ANOVA (for≥3 groups)
followed by Newman-Keuls multiple comparisons test. A value of P< 0.05 was considered
significant.
3. Results
3.1. Superoxide levels in LPS treated mouse lungs
Relative superoxide levels were determined by EPR in lung tissues harvested from 0- (Control),
2-, 4-, and 12-h after LP exposure (Fig. 1). Our data indicate that lung superoxide levels were
significantly increased early after LPS-exposure: 2 h (~2-fold) and 4 h (~1.5 fold) and that this
was a transient event as there was no change in superoxide levels 12 h post-LPS (Fig. 1). To
determine the contribution of uncoupled eNOS in the increased superoxide generation,
duplicate samples were incubated with the NOS inhibitor, NG-monomethyl L-arginine (L-
NMMA; 100 μM, 30 min) on ice prior to the addition of CMH. The LPS-mediated increase in
superoxide generation was blocked in the presence of L-NMMA suggesting that NOS
uncoupling is a significant contributor to superoxide generation after LPS exposure (Fig. 1).
Specificity of the EPR assay for superoxide was confirmed by a significant reduction in the
waveform amplitude with the addition of superoxide scavenger, polyethylene glycolconjugated
superoxide dismutase (PEG-SOD) to the samples (Fig. 1).
3.2. NOx and BH4 levels after LPS exposure
NOx levels were determined in mouse lung tissue 0-, 2-, 4-, and 12-h after LPS treatment.
Correlating with the increase in NOS uncoupling, we found there was a significant decrease
in NOx levels 2 h (−40%) after LPS exposure whereas we found an increase in NOx levels 4 h
(+60%) and 12 h (+160%) after LPS administration (Fig. 2 A). In addition, we measured lung
BH4 levels after LPS exposure. BH4 levels were unaltered 2 h after LPS exposure but were
significantly elevated at 4 h (~2 fold) and 12 h (~3 fold) post LPS treatment (Fig. 2 B).
3.3. NOS expression in control and ALI mouse lungs
To attempt to correlate the changes in NOx levels with NOS isoform protein levels, whole lung
homogenates were assessed for changes in the expression of NOS isoforms (eNOS, nNOS and
iNOS) 2- and 4-h after LPS exposure. Western blot analyses showed that there was no
difference in eNOS (Fig. 3 A) or nNOS protein levels (Fig. 3 B). However, iNOS protein levels
although unchanged 2 h post-LPS treatment were significantly increased (~6-fold) 4 h after
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7. LPS treatment (Fig. 3C) suggesting the increase in NOx levels 4 h post-LPS is likely due to
increases in iNOS protein levels.
3.4. Elevated ADMA levels and decreased DDAH activity in LPS-treated mouse lungs
In an attempt to evaluate the mechanism responsible for the increase in NOS uncoupling early
after LPS exposure, we next determined if LPS altered the levels of ADMA in the mouse lung.
We found significantly increased ADMA levels in LPS treated mouse lungs at 2 h (12.13 ±
0.84 vs. 7.53 ± 0.57 nmol/gww; Fig. 4 A), 4 h (13.40 ± 2.10 vs. 7.53 ± 0.57 nmol/gww; Fig.
4 A) and 12 h (19.10 ± 1.90 vs. 7.53 ± 0.57 nmol/gww; Fig. 4 A) after LPS exposure. To
determine whether the increased ADMA levels in the LPS treated mouse lungs were a result
of decreased DDAH I and/or DDAH II protein expression, we measured protein levels of the
two isoforms by Western blot analyses. DDAH I and DDAH II anti-serum detected ~37-kDA
and ~33-kDA bands, respectively in lung tissue homogenates. No significant differences were
detected between protein levels of either DDAH I (Fig. 4 B) or DDAH II (Fig. 4 C) in mouse
lung tissue post-LPS. However, we found that DDAH enzyme activity was significantly
decreased (~2-fold) in mouse lung tissue homogenates both 2- and 4-h after LPS exposure (Fig.
4 D).
3.5. Elevated nitrotyrosine levels after LPS exposure
Superoxides react with NO to form peroxynitrite that can modify proteins by interacting with
and nitrating tyrosine residues to form 3-NT. To determine the presence of tyrosine-nitrated
proteins in the mouse lungs after LPS exposure, the levels of 3-NT were assessed by Western
blotting to detect nitrated proteins. The 3-NT levels were quantified by obtaining the
densitometric units of all nitrated proteins (Fig. 5 A). Our data indicate that LPS exposure
significantly increases 3-NT levels 4 h after LPS-treatment in the mouse lung (Fig. 5 B).
3.6. Peroxynitrite scavengers cause a reduction in nitrated proteins after LPS exposure
To determine the effect of peroxynitrite scavenging on LPS-mediated 3-NT levels, we used
two potent peroxynitrite scavengers, MnTymPyp and uric acid. The animals were treated with
peroxynitrite scavengers prior to LPS exposure and 3-NT levels were determined 4 h post-
LPS. Both, MnTymPyp and uric acid significantly attenuated the LPS-induced increase in 3-
NT levels (Fig. 6 A).
3.7. Peroxynitrite scavengers attenuate the LPS mediated increase in lung permeability
The increase in lung permeability in response to LPS was determined by measuring the Evan
Blue dye leak, 12 h after LPS challenge. Our data indicate that there was a significant increase
in the lung leak in the LPS treated mice (~1.7 fold) while this increase in lung leak was
significantly reduced in the animals pre-treated with the peroxynitrite scavengers (MnTymPyp
and uric acid) (Fig. 6 B).
3.8. ADMA potentiates VEGF-induced decrease in endothelial barrier function
To determine if increases in ADMA alone are sufficient to induce endothelial cell barrier
disruption, HLMVEC were exposed or not to ADMA (5 μM) in the presence or absence of
VEGF (100 ng) and the effect on endothelial barrier function estimated by measuring changes
in transendothelial resistance (TER) using an ECIS apparatus. Our data indicate that ADMA
alone is not sufficient to induce barrier disruption but it does potentiate the VEGF-mediated
reduction in TER (Fig. 7).
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8. 4. Discussion
This study provides insight into a novel mechanism by which ADMA mediated nitrosative
damage may cause a loss of lung function in an LPS-induced mouse model of ALI. We found:
(1) decreased DDAH activity, which was correlated with increased ADMA levels; (2)
increased NOS uncoupling at early stages and increased iNOS expression at later stages of the
pathogenesis; (3) peroxynitrite mediated increase in 3-NT levels and subsequent increase in
lung permeability; (4) reduced 3-NT levels and decreased lung leak in mice receiving
peroxynitrite scavengers before endotoxin exposure. Interestingly our data also indicate that
alone increases in ADMA levels do not cause endothelial barrier disruption in vitro using
HLMVEC. However, ADMA does potentiate VEGF-induced barrier disruption suggesting
ADMA may be necessary but not sufficient to induce ALI. Thus, ADMA may act in concert
with other proteins to produce endothelial barrier dysfunction. Indeed a recent study has shown
that the ADMA/DDAH pathway regulates pulmonary endothelial barrier function through the
modulation of Rac1 signaling (Wojciak-Stothard et al., 2009). Further studies will be required
to elucidate the mechanism and key targets involved in ADMA mediated EC barrier disruption.
We, and others, have previously shown that increased levels of the endogenous NOS inhibitor,
ADMA can cause uncoupling of NOS and increased production of both reactive oxygen species
(ROS) and reactive nitrogen species (RNS) (Sud et al., 2008; Boger et al., 2000), which results
in oxidative and nitrosative stress in the cell. There is growing evidence that increased ADMA
levels are involved in the pathogenesis of a number of cardiovascular diseases (Miyazaki et
al., 1999; Takiuchi et al., 2004; Boger, 2003c; Bae et al., 2005). Further, ADMA has been
shown to cause increased peroxynitrite generation, and increased nitration events leading to
pathological conditions (Sud et al., 2008). However, the role of ADMA in ALI has not been
clarified. In this study, we demonstrate that lung tissue ADMA levels were significantly
increased within 2 h of LPS exposure suggesting that this is an early event in the pathogenesis
of the disease. ADMA is degraded through active metabolism by the enzyme, DDAH. DDAH
has two isoforms and it has been previously shown that mRNA (Tran et al., 2000) and protein
(Arrigoni et al., 2003) for both DDAH isoforms are expressed in the lung. We observed a
decrease in DDAH activity in ALI lung tissue, however our Western blot analyses did not
detect alterations in either DDAH I or DDAH II protein levels. This suggests that the decrease
in DDAH activity is not due to altered protein levels but is perhaps due to a post-translational
modification. However, further studies will be necessary to elucidate the mechanism by which
LPS regulates DDAH activity. Consistent with our results other studies have shown that
reduced DDAH activity, but not expression, is responsible for the plasma ADMA elevation in
hypercholesterolemia and hyperhomocysteinemia (Boger et al., 1998; Lin et al., 2002;
Stuhlinger et al., 2001). It has also been shown that the treatment of mice with a DDAH inhibitor
can cause higher plasma and blood vessel concentrations of ADMA (Leiper et al., 2007). Our
results are also consistent with a recent study in which LPS significantly increased the levels
of ADMA, decreased DDAH activity, and increased intracellular ROS production in human
endothelial cells (Xin et al., 2007). While prior studies have reported elevated ADMA levels
in patients with septic shock (O'Dwyer et al., 2006) and found that serum ADMA was
associated with increased vascular superoxide generation and eNOS uncoupling in human
atherosclerosis (Antoniades et al., 2009). Our data also indicate that the NOS uncoupling is a
transient phenomenon after LPS exposure and as all three NOS isoforms are present our data
cannot determine if one isoform predominates. NOS uncoupling is a complex process that can
be induced by a variety of conditions including increases in ADMA (Sud et al., 2008), decreases
in the substrate L-arginine (Settergren et al., 2009), and decreases in the NOS cofactor, BH4
(Bevers et al., 2006; Cai et al., 2005). We found that there was no change in lung BH4 levels
after 2 h LPS exposure but there was a substantial increase after 4- and 12-h. This suggests
that the increase in BH4 at the later time points may be able to overcome the uncoupling effect
of the increases in ADMA. Although this is speculative our BH4 data are in agreement with a
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9. prior study that reported increases in BH4 levels in the rat in both the plasma and tissues 3 h
after LPS exposure (Hattori et al., 1996).
Tissue NOx levels, a stable metabolite of NO, were significantly decreased 2 h after LPS
exposure but were markedly elevated 4- and 12-h post-LPS treatment. The decreased NOx
levels 2 h post-LPS were not due to decreased NOS protein levels but rather were due to
increased NOS uncoupling. NO is formed from arginine by the enzyme NO synthase with three
known isoforms: eNOS, nNOS, and iNOS which contribute to total NOS activity. eNOS and
nNOS are the two constitutive forms, whereas iNOS is induced by cytokines and bacterial
products (Szabo et al., 1995; Minc-Golomb et al., 1994). Previously, LPS treatment has been
shown to reduce eNOS protein and mRNA expression in bovine endothelial cells (Lu et al.,
1996) and a decrease in eNOS protein expression 12 h post-LPS in the mouse lung has been
suggested (Chatterjee et al., 2008). Studies have also found increased lung NO production
associated with increased iNOS expression and/or iNOS activity in various models of ALI
(Webert et al., 2000; Farley et al., 2006; Razavi et al., 2004; Scumpia et al., 2002; Okamoto et
al., 2000). Interestingly, in our studies, although increased NO production assessed indirectly
by measuring the levels of the oxidative metabolites of NO (NOx levels) at 4- and 12-h post-
LPS exposure, is associated with increased iNOS expression, we did not find any change in
eNOS and nNOS protein levels, suggesting that the increased bioavailable NO may be iNOS
derived. It is worth noting that some studies have indicated that nNOS also may be induced in
pathological conditions (Gocan et al., 2000) and that nNOS-derived NO may be involved in
the pathogenesis of ALI in sheep with sepsis (Enkhbaatar et al., 2003). These conflicting reports
suggest that studies using selective NOS inhibitors and investigating the individual roles of
eNOS, nNOS and iNOS on NO generation may be warranted.
NO and superoxide radicals can combine to form the toxic product, ONOO–, a potent RNS
which causes the irreversible nitration of tyrosine residues within proteins leading to cellular
damage and cytotoxicity. Increasing evidence suggests that peroxynitrite is involved in the
pathogenesis of endotoxin-induced endothelial injury, multiple organ dysfunction, and
hemodynamic instability (Salvemini et al., 2006; Cuzzocrea et al., 2006; Zingarelli et al.,
1997; Beckman, 2002). The levels of nitrated proteins have been shown to be significantly
elevated during inflammatory diseases in humans, including ALI (Haddad et al., 1994; Zhu et
al., 2001; Lamb et al., 1999; Salvemini et al., 2006; Cuzzocrea et al., 2006). In addition, a
recent study has shown evidence that immunoglobulins against tyrosine-nitrated proteins are
present in plasma of patients with ALI (Thomson et al., 2007) while increased iNOS expression
has been previously associated with increased 3-NT concentrations in the lungs of animal with
ALI (Peng et al., 2005; Mehta, 2005). Our previous in vitro studies have also shown that ADMA
uncouples eNOS leading to an increased peroxynitrite generation and subsequent elevation in
3-NT protein levels (Sud et al., 2008). In the present study, we found an almost 2-fold increase
in the 3-NT levels 4 h after LPS exposure and while the increases in 3-NT levels were blocked
by the addition of the ONOO– scavenger, uric acid and the superoxide scavenger, MnTymPyp.
Both scavenging ONOO– directly, by uric acid, or indirectly by decreasing superoxide levels,
with MnTymPyp, decreased total nitrated proteins and the lung permeability associated with
LPS exposure. Thus, our data along with the findings of previous studies suggest that that
blocking protein nitration events may be a potential therapeutic target for ALI. However, to
allow more specific therapies to be developed further studies will be required to identify the
relevant nitrated proteins and determine how nitration modulates their activity leading to
endothelial barrier disruption and ALI.
In conclusion, in this study we found a significant role for the ADMA/DDAH pathway in the
development of ALI. Increased ADMA levels lead to peroxynitrite mediated nitrosative
damage of proteins as indicated by increased 3-NT levels. Peroxynitrite scavengers reduced
the nitrated protein levels and decreased permeability events associated with ALI. Given
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10. increasing evidences of the role of ADMA in the pathogenesis of ALI, it may be a target for
pharmacotherapeutic interventions in patients with ALI.
Acknowledgments
This research was supported in part by grants HL60190 (SMB), HL67841 (SMB), HL084739 (to SMB),
R21HD057406 (to SMB), and HL61284 (to JRF) all from the National Institutes of Health, 0550133Z from the
American Heart Association, Pacific Mountain Affiliates (SMB), 09BGIA2310050 from the Southeast Affiliates (to
SS), and by a Transatlantic Network Development Grant from the Fondation Leducq (to SMB & JRF). This work was
also supported by a Programmatic Development award (to SMB, JC, and DF) and Seed Awards (to SS and SJ) from
the Cardiovascular Discovery Institute of the Medical College of Georgia. Anita Smith was supported in part by NIH
training Grant 5T32HL06699.
Abbreviations
ALI acute lung injury
LPS lipopolysaccharide
ADMA asymmetric dimethylarginine
DDAH dimethylarginine dimethylaminohydrolase
3-NT 3-nitrotyrosine
NO nitric oxide
NOS nitric oxide synthase
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15. Fig. 1.
Superoxide levels in the mouse lung after LPS exposure. Relative superoxide levels were
determined by electron paramagnetic resonance (EPR) in LPS-treated mouse lungs. There is
a significant increase in superoxide radical generation 2- and 4 h after LPS exposure that is
blocked by the addition of the NOS inhibitor, NG-monomethyl L-arginine (L-NMMA; 100
μM) or polyethylene glycol-superoxide dismutase (PEGSOD; 100 U/ml). There is no change
in superoxide generation 12 h after LPS exposure. Values are means ± SE, N=6. *P<0.05 vs.
no LPS, †P<0.05 vs. LPS alone.
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16. Fig. 2.
Lung tissue NOx and tetrahydrobiopterin levels after LPS exposure. Tissue NOx levels are
significantly decreased 2 h after LPS exposure. However, NOx levels were significantly
increased 4- and 12-h after LPS exposure (A). Lung BH4 levels were measured by HPLC.
There was no change in BH4 levels 2 h after LPS exposure, but BH4 levels were significantly
increased 4- and 12-h post LPS treatment (B). Values are mean±SE, N=5 for each group.
*P<0.05 compared to no LPS, †P<0.05 compared to previous time point.
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17. Fig. 3.
NOS isoform protein levels in the mouse lung after LPS exposure. Protein levels for eNOS,
nNOS, and iNOS were determined in lung tissues 2- and 4-h after LPS exposure by Western
blot analysis using specific antisera raised against eNOS, nNOS, or iNOS respectively and re-
probed with β-actin to normalize for loading. Representative Western blots are shown for eNOS
(panel A), nNOS (panel B), and iNOS (panel C). There are no changes in eNOS or nNOS
protein levels but iNOS protein levels are significantly increased 4 h after LPS exposure. Values
are mean ± SE, N=5 for each group *P<0.05 compared to no LPS.
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18. Fig. 4.
LPS exposure leads to elevated ADMA and decreased DDAH activity in the mouse lung.
ADMA levels and DDAH activity were analyzed by high-performance liquid chromatography
(HPLC) in the mice lungs after LPS exposure. There was a progressive increase in ADMA
levels after 2-, 4- and 12-h LPS exposure (A). Although there were no changes in either DDAH
I (B) or DDAH II (C) protein levels there was a significant decrease in DDAH activity 2- and
4-h after LPS exposure (panel D). Values are mean ± SE, N=5 for each group, *P<0.05
compared to no LPS.
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19. Fig. 5.
Nitrated protein levels are increased in the mouse lung after LPS exposure. Protein levels for
total nitrated proteins were determined in the mouse lung 4 h after LPS exposure. Homogenates
(25 μg) were separated on a 4–20% denaturing polyacrylamide gel, electrophoretically
transferred to PVDF-nitrocellulose membranes, and analyzed using specific antiserum raised
against 3-NT residues. A representative image for the Western blot analysis for 3-NT protein
levels is shown (A). The boxed area shows the region of total nitrated proteins that was used
to quantify 3-NT levels. Densitometric analysis indicates that LPS exposure increases total
nitrated proteins in the mouse lung (B). Values are mean ± SE, N=5. *P<0.05 vs. no LPS.
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20. Fig. 6.
Peroxynitrite scavenging decreases the LPS-mediated hyperpermeability in the mouse lung.
Protein levels for total nitrated proteins were determined in the mouse lung 4 h after LPS
exposure in the presence and absence of the ONOO– scavengers, uric acid and MnTymPyp.
The presence of either uric acid and MnTymPyp significantly decreased total nitrated protein
levels compared to that obtained with LPS alone (A) and this correlated with a significant
decrease in Evans blue dye levels in the lungs indicating a decrease in lung permeability (B).
Values are mean ± SE, N=5. *P<0.05 vs. no LPS, †P<0.05 vs. LPS alone.
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21. Fig. 7.
ADMA potentiates the decrease in transendothelial electrical resistance (TER) associated with
VEGF exposure in human lung microvascualr endothelial cells. Confluent were exposed or
not to ADMA (5 μM, 1 h). Cells were then exposed or not to VEGF (100 ng) and changes in
TER across the endothelial cell monolayer were measured by ECIS. Although ADMA alone
does not induce a change in TER there is a potentiation in the decrease in TER associated with
VEGF exposure. Values are mean ± SE, N=5. *P<0.05 vs. untreated, †P<0.05 vs. VEGF alone.
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