1985 — 1991 |
Schumacker, Paul T. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R55Activity Code Description: Undocumented code - click on the grant title for more information. |
Mechanisms For a Dependence of O2 Uptake On Delivery
Patients with acute hypoxemic respiratory failure associated with severe non-cardiogenic pulmonary edema exhibit a depressed ability to extract oxygen from the periphery. In these patients, a change in oxygen delivery (QO2 = cardiac output x arterial O2 content) caused by a change in cardiac output (Qt) is associated with a corresponding change in O2 uptake (VO2). With these changes in QO2 and VO2, mixed venous PO2 remains relatively constant at normal or high levels. By contrast, critically ill patients without respiratory failure show no dependence of VO2 on QO2 unless delivery falls below 330 ml/min/m2. An inability of peripheral tissues to extract sufficient oxygen to maintain aerobic metabolism may be caused by molecular diffusion limitation at the capillary level, due to an enhanced diffusion distance. Additionally, inefficient distribution of peripheral blood flow with overperfusion of low O2 extracting vessels and underperfusion of capillaries with high O2 uptake might explain an abnormal dependence of VO2 on QO2. Experimental studies are proposed to test the effects of increased or decreased hemoglobin P50 on the critical QO2 required for aerobic metabolism. Another group will clarify the relative contributions of altered P50 (Bohr effect) versus low pH in the enhanced peripheral O2 extraction efficiency reported during metabolic acidosis. Additional studies will test the influence of arteriolar tone on maintenance of VO2 by measuring critical QO2 and extraction during phenoxybenzamine blockade, hydralazine or low dose norepinephrine infusion. Other studies will examine the effects of intravascular coagulation or sepsis on the critical QO2 and extraction. A separate group will test the possibility that mild hypothermia may reduce VO2 to a greater extent than it lowers QO2, thereby providing a clinical tool for management of patients with increased critical QO2 and severe hypoxemia. Critical QO2 will be determined in each of these groups by measuring VO2 and lactic acid as QO2 is gradually reduced by plasmapheresis. Critical QO2 is determined in each animal from the QO2 below which VO2 is reduced. These studies will help to clarify the pathophysiological mechanisms responsible for a dependence of VO2 on QO2, and will help to uncover potentially therapeutic interventions for the maintenance of peripheral O2 extraction.
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1 |
1986 — 1990 |
Schumacker, Paul T. |
K04Activity Code Description: Undocumented code - click on the grant title for more information. |
Oxygen Transport in Respiratory Failure
An inability to oxygenate blood adequately in the lungs, and an inability of peripheral tissues to extract O2 from blood efficiently contribute to both the morbidity and mortality of acute respiratory failure. These studies aim to extend our understanding of mechanisms which limit both pulmonary and systemic O2 transport in acute respiratory failure. Laboratory studies of peripheral O2 extraction are proposed to (1) clarify effects of bacterial sepsis on O2 extraction; (2) quantify the relative contributions of microembolization, altered vascular reactivity and histotoxic effects to the abnormal relationship between O2 delivery (QO2) and uptake (VO2) in sepsis; (3) determine whether systemic microembolization can interfere with tissue O2 extraction and whether this is reversible; (4) clarify contributions of capillary derecruitment versus capillary O2 extraction limitation in setting the minimum O2 delivery required to maintain aerobic metabolism (QO2c); (5) clarify the value of vasoactive drugs or altered hemoglobin P50 in lowering the minimum QO2 required to maintain VO2. Clinical studies of peripheral O2 extraction will determine (1) whether patients with acute respiratory failure associated with septicemia demonstrate an abnormal relationship between O2 delivery and uptake; (2) whether patients with localized lung injury (aspiration pneumonia) demonstrate the same abnormal O2 extraction capacity. Laboratory studies of pulmonary O2 exchange are proposed to explore mechanisms and potential therapeutic value of new forms of ventilatory management of acute respiratory failure. Specifically I will study (1) effects of continuous flow ventilation (CFV) on efficiency of gas exchange assessed by multiple inert gas elimination in normal animals and experimental models of pulmonary edema or increased bronchomotor tone; (2) the potential value of combining CFV with IPPV in maintaining gas exchange in these models; (3) the value of high frequency ventilation combined with IPPV in maintaining gas exchange in these models. Clinical studies using multiple inert gas elimination will assess (1) advanced stages of ARDS, when edema and shunt have attenuated, but where V/Q inequality may still be present. The reason for sensitivity of arterial PO2 to altered FIO2 in this stage will clarify the respective contributions of V/Q inequality, mixed venous PO2 cardiac output, and sensitivity of the distribution to altered FIO2; (2) effects of aminophylline and increased FIO2 on gas exchange in patients with acute on chronic respiratory failure. Collectively, these studies will provide understanding of mechanisms that contribute to limitations in O2 transport to the mitochondria, from which may arise new insight into potentially therapeutic approaches.
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1 |
1992 — 1996 |
Schumacker, Paul T. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms For a Dependence of 02 Uptake On Delivery |
1 |
1992 — 1993 |
Schumacker, Paul T. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Microvascular Control of 02 Extraction in Hypoxia
As the O2 delivery (blood flow X CaO2) to a tissue is reduced, the O2 extraction ration increases to maintain tissue metabolism. Below a critical delivery, increases in O2 extraction are inadequate to maintain O2 uptake VO2), and further decreases in delivery are associated with O2 supply-dependent VO2. At the critical point, healthy tissues typically exhibit extractions of 60-75%, whereas patients with Adult Respiratory Distress Syndrome appear to extract less than 40%. Possible mechanisms underlying the O2 supply-limitation at the critical point include (a) the extent of perfused capillary density; (b) heterogeneity of capillary or conducting vessel transit times with respect to VO2; (c) gas transport resistance introduced by plasma surrounding red cells; (d) finite rate of oxygen offloading from hemoglobin; and (e) functional shunting of O2 within tissues. A major focus of this project is to clarify the roles of perfused capillary density and microvascular transit time heterogeneity in determining the level of tissue O2 extraction at the onset of supply-dependent VO2. Specific Aim I will measure capillary recruitment and microvascular transit time heterogeneity during progressive reductions in O2 delivery produced by lowering arterial PO2 (hypoxic hypoxia) or lowering blood flow (stagnant hypoxia) in normal isolated intestine and heart. These studies will test the hypothesis that differential adjustments in perfused capillary density or transit time heterogeneity can explain the observation that a similar critical O2 delivery is reached when delivery is reduced by progressive stagnant, hypoxic, or anemic hypoxia, despite widely differing venous O2 tensions at the critical point. Specific Aim II will quantify the significance of microvascular adjustments in perfused capillary density and transit time heterogeneity for O2 exchange, by measuring the critical point during pharmacologic vasodilation and vasoconstriction in isolated intestine and heart. Additional studies will determine the significance of perfused capillary density for critical O2 extraction by reducing capillary density using graded microembolization. Perfused capillary density will be measured independently using indicator dilution methodology and quantitative morphology. Indicator dilution data will be analyzed by two independent approaches, which account for the transit time heterogeneity and the return of interstitial tracer to the capillary. Morphological analysis will use colloidal carbon to identify perfused vessels, and will quantify transit time heterogeneity using measurements of local blood flows and vascular volumes. This work will clarify and quantify the relationships among capillary surface area, microvascular flow heterogeneity, and the ability of tissues to maintain supply-independent VO2 in the face of limited O2 delivery.
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1 |
1994 — 1995 |
Schumacker, Paul T. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Microvascular Control of 0xygen Extraction in Hypoxia
As the O2 delivery (blood flow X CaO2) to a tissue is reduced, the O2 extraction ration increases to maintain tissue metabolism. Below a critical delivery, increases in O2 extraction are inadequate to maintain O2 uptake VO2), and further decreases in delivery are associated with O2 supply-dependent VO2. At the critical point, healthy tissues typically exhibit extractions of 60-75%, whereas patients with Adult Respiratory Distress Syndrome appear to extract less than 40%. Possible mechanisms underlying the O2 supply-limitation at the critical point include (a) the extent of perfused capillary density; (b) heterogeneity of capillary or conducting vessel transit times with respect to VO2; (c) gas transport resistance introduced by plasma surrounding red cells; (d) finite rate of oxygen offloading from hemoglobin; and (e) functional shunting of O2 within tissues. A major focus of this project is to clarify the roles of perfused capillary density and microvascular transit time heterogeneity in determining the level of tissue O2 extraction at the onset of supply-dependent VO2. Specific Aim I will measure capillary recruitment and microvascular transit time heterogeneity during progressive reductions in O2 delivery produced by lowering arterial PO2 (hypoxic hypoxia) or lowering blood flow (stagnant hypoxia) in normal isolated intestine and heart. These studies will test the hypothesis that differential adjustments in perfused capillary density or transit time heterogeneity can explain the observation that a similar critical O2 delivery is reached when delivery is reduced by progressive stagnant, hypoxic, or anemic hypoxia, despite widely differing venous O2 tensions at the critical point. Specific Aim II will quantify the significance of microvascular adjustments in perfused capillary density and transit time heterogeneity for O2 exchange, by measuring the critical point during pharmacologic vasodilation and vasoconstriction in isolated intestine and heart. Additional studies will determine the significance of perfused capillary density for critical O2 extraction by reducing capillary density using graded microembolization. Perfused capillary density will be measured independently using indicator dilution methodology and quantitative morphology. Indicator dilution data will be analyzed by two independent approaches, which account for the transit time heterogeneity and the return of interstitial tracer to the capillary. Morphological analysis will use colloidal carbon to identify perfused vessels, and will quantify transit time heterogeneity using measurements of local blood flows and vascular volumes. This work will clarify and quantify the relationships among capillary surface area, microvascular flow heterogeneity, and the ability of tissues to maintain supply-independent VO2 in the face of limited O2 delivery.
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1 |
1996 — 2006 |
Schumacker, Paul T. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Respiratory and Metabolic Adaptation to Cellular Hypoxia @ Northwestern University
DESCRIPTION (provided by applicant): Mammalian cells respond to low 02 levels (hypoxia; P02=3-30 mm Hg) by activating adaptive responses that help to restore 02 supply and prevent hypoxic injury. Controversy exists regarding the identity of the cellular 02 sensor triggering these responses. Our studies have revealed that mitochondria act as 02 sensors by increasing their release of reactive oxygen species (ROS) during hypoxia. These ROS function as early signals in the pathway linking the 02 sensor to the downstream responses, which include the activation of the Hypoxia-Inducible Factor-1 (HIF-1). HIF-1 activation triggers the increased expression of glycolytic enzymes, glucose transporters, and other genes during hypoxia. This application proposes to test the hypothesis that mitochondria act as the 02 sensor responsible for activation of HIF-1 during hypoxia, by releasing ROS. Studies using pharmacological tools in the previous funding period implicated mitochondrial Complex Ill as the site of increased ROS production. To test this more definitively, in Aim 1 we will generate a targeted knockout of the Rieske iron-sulfur protein (RISP), a nuclear-encoded gene that is required for the generation of ROS at Complex III. In murine embryonic stem cells lacking this gene, we predict that hypoxic stabilization of HIF-1 will be lost, while responses to anoxia, cobalt and exogenous H202 will be retained. Recent studies indicate that the small GTPase racl is required for HIF-1 alpha stabilization during hypoxia. Rac1 may act by amplifying the mitochondrial ROS signal generated during hypoxia by engaging additional oxidase systems, by amplifying mitochondrial ROS generation, or by triggering the relocation of mitochondria toward the nucleus. Aim 2 will determine whether mitochondrial ROS signals activate rac- 1 during hypoxia, and whether rac 1 then promotes the stabilization of HIF-1 alpha by amplifying the ROS signal. We hypothesize that the activity of prolyl hydroxylase, the enzyme that targets the HIF-l alpha subunit for proteasomal degradation during normoxia, is not intrinsically an 02 sensor but rather is regulated by signals initiated by the mitochondria. Aim 3 will clarify the role of increased mitochondrial ROS signaling in the stabilization of HIF-1 alpha during hypoxia by studying the regulation of prolyl hydroxylase activity by mitochondrial ROS signals. This will be tested in cultured cells and in an in vitro system where hydroxylation of a recombinant protein is assessed. Collectively, these studies will provide a more conclusive test of the hypothesis that mitochondrial ROS are the site of 02 sensing responsible for HIF-1 activation and gene expression during hypoxia.
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1 |
1998 — 2001 |
Schumacker, Paul T. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Oxygen Sensing and Cell Signaling in Hypoxia
DESCRIPTION (Adapted from the applicant's abstract): Although cellular respiration is set by metabolic need during normoxia, evidence suggests that cells can adapt to hypoxia by reducing their energy demand, thereby lowering the use of ATP and the need for O2. At reoxygenation, normal metabolic processes are restored and cellular activity recovers. An ability to reduce energy demands in hypoxia while preserving high energy phosphate levels (hypoxic adaptation) may be protective during severe hypoxia by conserving ATP for essential processes. Hypoxic adaptation requires a cellular O2 sensor capable of detecting PO2. Although multiple sensors likely exist, data suggest that cytochrome oxidase acts as the sensor during hypoxic adaptation. Specific Aim 1 will test whether cytochrome oxidase functions as the O2 sensor during hypoxia by decreasing its apparent Vmax. This will be tested using inhibitors that reduce the Vmax of the oxidase during normoxia, determining whether these activate the hypoxic adaptation response. Activation of the O2 sensor during hypoxia must be coupled to subsequent activation of an intracellular signaling cascade, which ultimately inhibits ATP utilization. Specific Aim 2 will test the hypothesis that reactive oxygen species (ROS) function as a second messenger in this signaling pathway. The PO2-dependent ROS generation in intact cells will be studied and correlated with the function of the oxidase. Other studies will confirm whether mitochondria are the source of the ROS and will link these signals to the activation of the hypoxic adaptation response. Studies with isolated mitochondria will identify the sites and mechanisms of PO2-dependent ROS generation during hypoxia. Collectively, these studies will clarify the mechanisms of mitochondrial ROS generation during hypoxia and link these signals to the function of cytochrome oxidase and the hypoxic adaptation response. The hypothesis is that signaling elements downstream of ROS lead to inhibition of ATP-dependent enzyme systems. Specific Aim 3 will begin to test the hypothesis that protein kinases function as downstream signaling elements in the hypoxic response. The involvement of protein kinase C in this pathway will be tested, based on previous studies demonstrating its activation by ROS or by hypoxia. The long term goal of this project is to identify O2 sensing mechanisms and the downstream signaling sequence involved in hypoxic adaptation. These studies will identify a novel pathway of cellular O2 detection, and may help clarify understanding of how cells adapt to lowered O2 conditions.
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1 |
2000 — 2003 |
Schumacker, Paul T. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
O2 Sensing by Mitochondria During Intermittent Hypoxia
Obstructive sleep apnea (OSA) is a clinical syndrome characterized by repeated episodes of severe hypoxemia caused by intermittent closure of the upper airway during sleep. Complications of OSA include pulmonary hypertension caused by vascular remodeling in the lung. Repeated intermittent hypoxia is the most likely cause of this remodeling. The remodeling responses to hypoxia imply that a cellular O2 sensor exists that is capable of responding to rapid changes in [O2]. Studies from this laboratory indicate that mitochondria function as O2 sensors during hypoxia in diverse cells, releasing reactive oxygen species (ROS) to the cytoplasm that trigger intracellular signaling pathways leading to the activation of the transcription factors Nuclear Factor kappa B (NFkB) and Hypoxia-Inducible Factor (HIF- 1) in some cells, and that mediate adaptive metabolic responses in others. This application proposes that mitochondria also function as O2 sensors during intermittent hypoxia, by releasing ROS that lead to the activation of the transcription factors NFkB, HIF- I and AP- I that regulate genes involved in long-term vascular remodeling. Growth factors contribute to proliferation of cells in the vascular wall, and hypoxia amplifies their mitogenic response via an unknown mechanism. Mitochondrial ROS released during hypoxia could amplify the mitogenic response to growth factors by augmenting the oxidant signaling required for their proliferative response. Aim I will determine whether mitochondria function as O2 sensors by releasing ROS during intermittent hypoxia. Aim 2 will determine whether these ROS are necessary and sufficient for the activation of the transcription factors NFkB, HIF- I and AP- 1, and whether these factors mediate the subsequent transcriptional activation of target genes involved in vascular remodeling. Aim 3 will determine whether intermittent hypoxia amplifies the proliferative response to mitogens by stimulating mitochondrial ROS generation that augments growth factor-induced non-mitochondrial oxidant signaling. Collectively, these studies could identify a novel mechanism of O2 sensing in the lung, and provide a mechanistic explanation for the activation of gene transcritpion and cellular proliferation during intermittent hypoxia.
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1 |
2005 — 2009 |
Schumacker, Paul T. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
O2 Sensing in Hypoxic Pulmonary Vasoconstriction @ Northwestern University At Chicago
DESCRIPTION (provided by applicant): Hypoxic pulmonary vasoconstriction (HPV) helps to optimize lung gas exchange, but it contributes to pulmonary hypertension in hypoxic lung disease. 2 opposing models have emerged to explain the underlying mechanism of O2 sensing in HPV. 1 proposes that hypoxia decreases reactive oxygen species (ROS) generation, shifting the cytosol to a more reduced state. The other proposes that hypoxia stimulates ROS, generating an oxidant signal in the cytosol. Resolution of this debate has been hindered by a lack of tools to assess intracellular redox. In Aim 1 we will use novel redox-dependant Fluorescence Resonance Energy Transfer (HSP-FRET) and RoGFP1 probes to assess redox in normoxic and hypoxic pulmonary vascular cells. We hypothesize that increased ROS come from the mitochondrial electron transport chain (ETC). We will target overexpression of antioxidant enzymes to mitochondrial matrix or the cytosol to determine which compartments participate in redox signaling. Aim 2 will determine which ETC complexes contribute to ROS generation by using short hairpin interfering RNA (shRNA) to suppress expression of critical ETC subunits. We predict that oxidant signals will be attenuated when the subunits required for ROS generation are suppressed. Aim 3 will test the relationship between mitochondrial ROS generation and functional responses to hypoxia in pulmonary artery (PA) myocytes (increase in cytosolic Ca2+) and in PA endothelial cells (increased activation of Hypoxia Inducible Factor-1 and increased expression of endothelin-1). We predict that inhibiting the propagation of ROS signals from mitochondria to cytosol (by targeted overexpression of antioxidant enzymes) or preventing their generation (by shRNA suppression of critical ETC subunits) will abrogate the functional responses to hypoxia in both cell types. Collectively, these studies will test whether a common O2 sensing mechanism functions in PA myocytes and endothelial cells to trigger their diverse responses in HPV.
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0.988 |
2009 — 2010 |
Schumacker, Paul T. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Monitoring Cellular Redox Signaling and Oxidant Stress in Vivo @ Northwestern University At Chicago
DESCRIPTION (provided by applicant): Healthy cells use low levels of reactive oxygen species (ROS) as second messengers in signal transduction pathways. High levels of ROS cause oxidative damage to proteins, lipids and DNA. Oxidant stress has been implicated in the cellular dysfunction associated with ischemia- reperfusion injury, vascular disease, stroke, diabetes, neurodegenerative diseases, liver disease, renal disease, inflammation, cancer, and other disorders. As the awareness of the role of redox stress in health and disease has grown, the demand for new tools to monitor oxidant stress in vivo has increased. Current methods to assess redox events are limited by their inability to provide quantitative data or spatial information on the subcellular sites of oxidant generation. Moreover, existing probes are generally unsuitable for in vivo studies. New methods to monitor intracellular oxidant stress in intact tissues could enhance our understanding of how cell-cell interactions and tissue microenvironments influence the generation of ROS. We propose to create a new system to detect redox status and oxidative stress in specific cells within intact tissues, using a novel combination of existing methods. In Aim 1 we will create transgenic mice with DNA encoding the redox-sensitive fluorescent protein, RoGFP, inserted at a LoxP-silenced ROSA26 genomic locus. Three lines will be generated, which target the RoGFP sensor to cytosol, mitochondrial matrix, or mitochondrial intermembrane space. In Aim 2 we will activate expression of the RoGFP genes in primary cells cultured from these mice, using Cre recombinase to delete the stop codon. We will confirm correct targeting of the expressed protein, and confirm its function in response to redox stress. In Aim 3 we will breed the RoGFP mice with smooth muscle-specific Cre recombinase mice, to elicit RoGFP expression in pulmonary artery smooth muscle cells in the lung. Using that model system to demonstrate efficacy, we will measure redox changes in smooth muscle cells in the intact lung during ventilation with different concentrations of oxygen. Two-photon microscopy will be used to assess the redox status of the subcellular targeted RoGFP proteins in vivo. These animals will therefore provide exciting new tools that will enable us, and other investigators, to monitor subcellular oxidative stress in intact tissue in diverse cell types and disease models. Public Health Relevance Statement: Healthy cells in the body use oxygen free radicals (Reactive Oxygen Species, or ROS) to regulate various cellular functions. Excessive levels of ROS disrupt cell function, and they contribute to cellular injury in a large number of diseases. To understand how ROS affect cells, it is essential to monitor their levels. However, current tools are limited in their ability to assess intracellular ROS. We propose to correct this problem by inserting a gene encoding an ROS-sensitive fluorescent protein into mice. When the gene is turned on, the cell will generate a protein that moves to a known intracellular compartment and signals a change in ROS levels by altering its fluorescence. We will turn this gene on in certain types of cells in the mouse, and measure the fluorescence changes using a form of microscopy that can "see" deeply into intact tissues. We will test the performance of this sensor in the lungs, where we will measure the ROS response to changes in the concentration of oxygen that the animal is breathing. However, many other investigators will be able to use the same mice where, by turning on the reporter gene in other cell types, it will be possible to monitor ROS in a wide range of different tissues. Hence, this mouse will provide useful information on ROS levels in a wide range of disease models. The successful outcome of this project is supported by extensive preliminary studies demonstrating the feasibility of each step in the process. The end result should significantly extend our ability to assess ROS in intact tissues, in animal models of disease.
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0.988 |
2009 — 2018 |
Schumacker, Paul T. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Respiratory and Metabolic Adaptation to Hypoxia @ Northwestern University At Chicago
DESCRIPTION (provided by applicant): Cardiovascular and pulmonary diseases can disrupt the supply of O2 to tissues. Inadequate O2 supply can threaten cell survival and trigger organ failure. All mammalian cells can detect decreases in oxygen availability (hypoxia), and activate adaptive responses that protect them from the consequences of O2 deprivation. However, the underlying mechanisms of hypoxia sensing are not known. We hypothesize that mitochondria function as hypoxia sensors in the cell. These organelles appear to trigger adaptive responses by initiating a signaling cascade involving an increased release of reactive oxygen species (ROS) from the electron transport chain. We propose to study the molecular mechanisms underlying this response, and the role of the mitochondrial hypoxia sensor in the regulation of adaptive responses to hypoxia. Aim 1 will test whether conformational changes in mitochondrial Complex III induced by hypoxia cause an increase in ROS release to the intermembrane space and the cytosol. These oxidants may trigger adaptive responses including the stabilization of Hypoxia-Inducible Factor-1 alpha (HIF-1a) and the activation of hypoxic pulmonary vasoconstriction. This aim will be tested by protein crystallization studies of intact Complex III under different O2 concentrations. We will study the requirement for Complex III in hypoxia sensing by employing a new mouse model with conditional deletion of RISP, a functional component of the Complex required for ROS generation. Aim 2 will test whether increased hypoxia-induced ROS release from Complex III leads to an increase in oxidant signaling in the mitochondrial intermembrane space and the cytosol. This will be tested using novel protein-based redox sensors targeted to subcellular compartments. Aim 3 will clarify the signaling pathways linking the increase in ROS signaling during hypoxia and the downstream stabilization of HIF-1a. We hypothesize that Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx) functions as a signal transduction messenger in the ROS-HIF-1a pathway, transmitting the hypoxia-induced ROS to the stabilization of HIF-1a. We will also test the hypothesis that oxidation of HIF-1a itself, through the redox modification of a cysteine thiol, contributes to the regulation of its stability. Excessive or inadequate activation of the hypoxia sensing pathway contributes to cardiovascular and pulmonary disease pathogenesis, justifying these studies in terms of clinical relevance. PUBLIC HEALTH RELEVANCE: The cells in the body use oxygen to generate energy. When diseases of the lungs, blood vessels or the heart interfere with the supply of oxygen to tissues, the cells of the affected tissues can fail to perform normally, or even die. Each cell in the body has the ability to sense how much oxygen it receives. When the supply of oxygen to a cell decreases, a sensor in the cell activates a set of adaptive responses that protect it from damage in the event that oxygen becomes critically limiting. In this regard, the cellular oxygen sensor is important for normal health. However, in some diseases excessive or insufficient activation of the oxygen sensor can contribute to the effects of the disease. The mechanism of cellular oxygen sensing is not understood. This project seeks to test the hypothesis that mitochondria, organelles in the cell that normally consume oxygen, are the site of oxygen sensing. We have developed a new mouse model to test this hypothesis, which allows us to inactivate the ability of mitochondria to generate oxygen free radicals. We hypothesize that free radicals generated by the mitochondria are responsible for activating the adaptive responses to low oxygen conditions. In particular, we will test the hypothesis that a protein termed HIF, which is responsible for activating protective genes in response to low oxygen, is controlled by the free radical signaling from the mitochondria. Collectively, these studies will provide new information regarding the mechanisms of oxygen sensing in cells, which is important in normal tissues and in disease states.
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0.988 |
2014 — 2017 |
Chandel, Navdeep S (co-PI) [⬀] Schumacker, Paul T. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Metabolic Regulation of Pulmonary Vascular Remodeling @ Northwestern University At Chicago
Pulmonary hypertension develops in patients with alveolar hypoxia arising from chronic lung diseases or high altitude exposure. These diseases affect large numbers of patients, and hypoxia-induced pulmonary vascular remodeling is the most common cause of pulmonary hypertension (PH). We propose that remodeling is triggered by mitochondrial O2 sensors that initiate redox signaling in vascular smooth muscle, thereby promoting cell contraction, growth and proliferation. Our previous work implicates mitochondria as a source of reactive oxygen species (ROS) signals that activate functional responses to acute hypoxia in pulmonary artery smooth muscle cells (PASMC). We now propose to test whether these signals also drive vascular remodeling and metabolic reprogramming in PASMC, leading to PH during chronic hypoxia. The role of ROS in PH has been highly controversial, so these studies are critically important for clarifying this issue. Chronic hypoxia activates redox signaling and induces Hypoxia-Inducible Factors (HIF-1 and HIF-2) in pulmonary vascular cells. This promotes metabolic reprogramming toward glycolysis and away from mitochondrial oxidative phosphorylation. We will test whether this reprogramming promotes vascular remodeling and PH. Hypoxia and ROS also activate AMP- dependent Protein Kinase (AMPK), a cellular energy sensor that activates catabolic pathways and inhibits anabolic metabolism, potentially opposing the remodeling response. We will determine whether AMPK activation can limit the growth, proliferation and metabolic reprogramming of PASMC, thereby opposing the development of PH. To achieve these aims we have assembled a powerful set of tools to quantify and modify redox signaling and metabolic pathways, which will be applied in genetic mouse models of PH and in pulmonary vascular cells from patients with pulmonary hypertension. These studies will provide novel insight into the mechanisms regulating the remodeling and PH arising in response to chronic hypoxia, and will identify potential therapeutic targets for the treatment of hypoxia-associated PH in humans.
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0.988 |
2018 |
Schumacker, Paul T |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Molecular Core @ Northwestern University At Chicago
Project Summary This Core will provide support to Projects 1, 2 and 3 by providing molecular tools to manipulate gene expression in the proposed studies, and by providing support for the cell-specific isolation of mRNA from target cell types, and for RT-PCR analysis of that RNA to assess changes in gene expression. Aim 1 will be to design, develop, and validate recombinant adeno-associated viral expression vectors (rAAVs) for distribution to Projects 1-3. These vectors will be used to express proteins under the control of tissue-specific promoters, either in vivo using stereotaxic injection, or in cultured iPSc-derived dopaminergic neurons. Aim 2 will be to design, develop and validate rAAVs to be used for delivery of shRNA gene knock down constructs for use in each of the Projects. Aim 3 will be to provide services involving cell-specific isolation of RNA from targeted cells using RiboTag-based isolation of mRNA, and to identify and quantify genes undergoing changes in expression/translation, by RT-PCR analysis of that mRNA. Core B team members are highly skilled in the development and use of these tools. Finally, this Core will support the Projects by developing novel molecular tools, should the need arise.
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0.988 |
2018 |
Schumacker, Paul T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Redox Regulation of Vascular Cgmp Signaling in Neonatal Lungs @ Northwestern University At Chicago
SUMMARY Bronchopulmonary dysplasia (BPD) is a common complication of preterm birth affecting 30% of infants with birthweights < 1000 grams. Recently, pulmonary hypertension (PH) and right ventricular hypertrophy (RVH) have been recognized as complications in approximately 25% of infants with moderate or severe BPD. Once infants develop PH, little is known about how to treat them, and risk of morbidity and mortality is very high. One of the mainstays of BPD therapy is oxygen (O2), but supraphysiologic O2 concentrations in combination with mechanical ventilation increase reactive oxygen species (ROS) production, inducing significant vascular dysfunction in neonates. Potential key targets for ROS-mediated dysregulation in the pulmonary vasculature are involved in cGMP signaling - soluble guanylate cyclase (sGC) and phosphodiesterase 5 (PDE5). In the previous funding period, we utilized a mouse model of hyperoxia-induced lung disease and PH to demonstrate that hyperoxia-exposed mice develop significant pulmonary and vascular disease, characterized by alveolar simplification, fewer capillaries, small pulmonary arteries (PA) remodeling, and RVH. We demonstrated that hyperoxia rapidly decreased lung and PA soluble guanylate cyclase (sGC) expression and activity and increased lung and PA phosphodiesterase 5 (PDE5) activity, leading to disruption of cGMP-mediated downstream signaling. Giving low-dose sildenafil, a PDE5 inhibitor, concurrent with hyperoxia prevented increased PDE5 activity, vascular remodeling, and RVH, but was unable to restore normal capillary density and alveolarization. In preliminary data for this proposal, we have demonstrated that another environmental stressor, intrauterine growth restriction (IUGR) due to placental insufficiency, leads to a significant delay in alveolarization with decreased expression of a key lung growth factor, insulin-like growth factor-1 (IGF-1), decreased sGC expression and activity, and impaired alveolarization. IUGR mice have an exaggerated phenotype with hyperoxia vs. appropriately grown mice with further decreased sGC expression and activity and impaired alveolarization. We hypothesize that both growth restriction and hyperoxia-induced mitochondrial ROS disrupt the critical sGC-cGMP signaling pathway, leading to impaired alveolarization and angiogenesis. We will utilize our established mouse model of hyperoxia-induced lung injury in combination with a novel model of IUGR to elucidate the molecular mechanism by which ROS and growth restriction disrupt sGC-cGMP signaling and lung development. These studies will provide the pathophysiologic, mechanistic framework to improve pharmacologic treatment of BPD infants with PH. We believe sGC is a key integrator for multiple signals that impact alveolarization and angiogenesis in the neonatal period. sGC stimulators such as riocinguat are approved in adults with PH and represent a novel and potentially immediate therapeutic option for BPD-PH infants if a rationale for their use can be demonstrated.
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0.988 |
2019 — 2020 |
Schumacker, Paul T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Redox Regulation in the Perinatal Pulmonary Vasculature @ Lurie Children's Hospital of Chicago
SUMMARY Bronchopulmonary dysplasia (BPD) is a common complication of preterm birth affecting 30% of infants with birthweights < 1000 grams. Recently, pulmonary hypertension (PH) and right ventricular hypertrophy (RVH) have been recognized as complications in approximately 25% of infants with moderate or severe BPD. Once infants develop PH, little is known about how to treat them, and risk of morbidity and mortality is very high. One of the mainstays of BPD therapy is oxygen (O2), but supraphysiologic O2 concentrations in combination with mechanical ventilation increase reactive oxygen species (ROS) production, inducing significant vascular dysfunction in neonates. Potential key targets for ROS-mediated dysregulation in the pulmonary vasculature are involved in cGMP signaling - soluble guanylate cyclase (sGC) and phosphodiesterase 5 (PDE5). In the previous funding period, we utilized a mouse model of hyperoxia-induced lung disease and PH to demonstrate that hyperoxia-exposed mice develop significant pulmonary and vascular disease, characterized by alveolar simplification, fewer capillaries, small pulmonary arteries (PA) remodeling, and RVH. We demonstrated that hyperoxia rapidly decreased lung and PA soluble guanylate cyclase (sGC) expression and activity and increased lung and PA phosphodiesterase 5 (PDE5) activity, leading to disruption of cGMP-mediated downstream signaling. Giving low-dose sildenafil, a PDE5 inhibitor, concurrent with hyperoxia prevented increased PDE5 activity, vascular remodeling, and RVH, but was unable to restore normal capillary density and alveolarization. In preliminary data for this proposal, we have demonstrated that another environmental stressor, intrauterine growth restriction (IUGR) due to placental insufficiency, leads to a significant delay in alveolarization with decreased expression of a key lung growth factor, insulin-like growth factor-1 (IGF-1), decreased sGC expression and activity, and impaired alveolarization. IUGR mice have an exaggerated phenotype with hyperoxia vs. appropriately grown mice with further decreased sGC expression and activity and impaired alveolarization. We hypothesize that both growth restriction and hyperoxia-induced mitochondrial ROS disrupt the critical sGC-cGMP signaling pathway, leading to impaired alveolarization and angiogenesis. We will utilize our established mouse model of hyperoxia-induced lung injury in combination with a novel model of IUGR to elucidate the molecular mechanism by which ROS and growth restriction disrupt sGC-cGMP signaling and lung development. These studies will provide the pathophysiologic, mechanistic framework to improve pharmacologic treatment of BPD infants with PH. We believe sGC is a key integrator for multiple signals that impact alveolarization and angiogenesis in the neonatal period. sGC stimulators such as riocinguat are approved in adults with PH and represent a novel and potentially immediate therapeutic option for BPD-PH infants if a rationale for their use can be demonstrated.
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