7/9/2008. Hypoxic cell injury. Consequences of hypoxia depend on cell type. Significance of hypoxia depends on:

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1 Cellular Adaptation and Cell Injury CLDavis Foundation On the Beach Lecture 2 Hypoxia, reperfusion, free radicals, and apoptosis R K Myers 2008 Hypoxic cell injury Hypoxia: any state of reduction of O 2 supplied to cells and tissues and results in decreased ATP production. Hypoxia can result from: cardiorespiratory failure loss of blood supply reduced transport of O2 in blood (anemia or CO toxicosis) blockage of cell enzymes (e.g. cyanide) Ischemia is decreased blood supply or perfusion of tissues usually due to constriction or obstruction of blood vessels. Ischemia tends to injure tissue faster because substrates for glycolysis are not delivered, anaerobic generation of ATP stops faster, glycolytic function is inhibited by metabolite accumulation. Consequences of hypoxia depend on cell type Highly vulnerable to hypoxia: Relatively resistant to hypoxia: cardiac myocytes Adipocytes neurons Bones proximal tubule epithelium Skin endothelial cells Muscle pneumocytes Significance of hypoxia depends on: Organ/Cell type Degree Duration Extent 1

2 Events in ischemia: reversible and irreversible injury Ischemia -- Reperfusion Injury When blood flow is restored to previously ischemic but not dead cells, injury may become worse and be accelerated. Loss of cells in addition to cells that are irreversibly damaged at the end of ischemia. Very important clinically in myocardial infarction and stroke. Ischemia -- Reperfusion Injury Reoxygenation by increased generation of free radicals Compromised cellular antioxidant defenses Promotion of the mitochondrial permeability transition precludes mitochondrial energization and ATP recovery Inflammation resulting from cytokines and increased adhesion molecules from parenchymal and endothelial cells. Leukocyte influxes adds to damage. Activation of complement pathway 2

3 Reperfusion injury Exuberant free radical formation occurs with reperfusion from parenchymal, endothelial cells, and leukocytes due to: Xanthine oxidase pathway Mitochondrial electron transport chain Conversion of NOS to produce superoxide (rather than NO) NADPH oxidase from infiltrating leukocytes Mark Ackermann, Iowa State U Free radical formation during reperfusion: xanthine oxidase Cell ischemia/loss of oxygen leads to ATP degradation. If perfusion is re-established, oxidative radicals are formed. ATP ISCHEMIA ADP Xanthine dehydrogenase AMP Adenosine Inosine Xanthine oxidase Hypoxanthine O2 O2 - REPERFUSION Xanthine oxidase Xanthine SOD H2O2 Fe++. OH Uric acid Tissue injury Mark Ackermann, Iowa State U Proximity of muscle and other cells to endothelial cells in reperfusion injury Direct contact between myofibers and other cells to endothelial cells allows passage of nucleotides via nucleotide transport proteins (NTP) that can be used in the xanthine oxidase pathway Muscle or other cell ATP-ADP-AMP-Adenosine-Inosine NTP NTP Adenosine-Inosine Endothelial cell X.O. pathway Mark Ackermann, Iowa State U

4 Mitochondrial activity contribution to ROS in reperfusion injury Distal to NADPH dehydrogenase in the mitochondrial electron transport chain Ubiquinone (CoQ) increases ROS formation ROS induce mitochondrial permeability transition (MPT) These contribute to ROS formation and reperfusion injury Mark Ackermann, Iowa State U Increased superoxide generation from NOS during reperfusion injury All three NOS (nnos, enos and inos) can switch from NO to superoxide generation This occurs with depletion of NOS substrate (arginine and BH4) Both arginine and BH4 are depleted by ROS (vicious cycle) The increased superoxide production contributes to ROS damage and reperfusion injury Mark Ackermann, Iowa State U Leukocyte infiltration with reperfusion injury Hypoxic tissues, including myocytes and endothelial cells increase expression of leukocyte adhesion molecules Enhanced leukocyte (neutrophil) infiltration with reperfusion Increased activity of NADPH oxidase by infiltrating leukocytes NADPH oxidase produces additional ROS Mark Ackermann, Iowa State U

5 Reperfusion injury therapy Therapies Needed to reduce ROS damage but yet allowing perfusion and return to normoxia Antioxidants Anti-neutrophil Cell Death by Apoptosis Apoptosis (apoptotic necrosis) a type of programmed cell death with initiation of a selfinduced cell death process ( cell suicide ) A variety of stimuli result in self-programmed, genetically determined, energy-dependent sequences of molecular events involving initiation by cell signaling, control and integration by regulatory molecules, a common execution phase by caspase family genes, and dead cell removal Cell Death by Apoptosis Initiation phase: caspases (cysteine proteases that cleave aspartic acid residues) become catalytically active by intrinsic and extrinsic paths. Execution phase: specific caspase enzymes act to cause cell death 5

6 Buds Blebs The sequential ultrastructural changes seen in necrosis (left) and apoptosis (right). In apoptosis, the initial changes consist of nuclear chromatin condensation and fragmentation, followed by cytoplasmic budding and phagocytosis of the extruded apoptotic bodies. Signs of cytoplasmic blebs, and digestion and leakage of cellular components characterize necrosis. (Adapted from Walker NI, Harmon BV, Gobe GC, Kerr JF: Methods Achiev Exp Pathol 13:18-54, 1988.)(McGavin, M. Donald. Pathologic Basis of Veterinary Disease, 4th Edition. C.V. Mosby, Morphologic features of oncotic and apoptotic cell death Oncotic Necrosis Morphology:.Groups of cells swell and then may shrink.pyknosis karyorrhexis karyolysis.cytoplasm is disrupted.cytoplasmic blebs form. Enzymatic digestion, contents may leak from cell.inflammation is frequent Invariably pathologic Apoptotic Cell Death Morphology:.Individual cells are shrunken..chromatin is condensed. Fragmentation into nucleosome size fragments (pyknosis and karyorrhexis).cytoplasm is fragmented, membranes intact.cytoplasmic buds form and may be found in adjacent cells and phagocytes as apoptotic bodies..inflammation is absent Can be physiologic or pathologic Causes of Apoptosis Normal occurrence in many situations, physiologic and pathologic Eliminates unwanted, potentially harmful, useless, damaged cells. DNA damage of many types is a cause, e.g. due to failure of DNA repair 6

7 Physiologic Apoptosis Programmed cell destruction during embryogenesis (implantation, organogenesis, developmental involution, metamorphosis. PCD Hormone dependent involution in adults. E.g. endometrial and uterine involution Cell deletion in proliferating cell populations. Skin, gut Death of inflammatory and immune cells Elimination of self reactive lymphocytes Cell death by cytotoxic T cells. Defense mechanism against viruses, tumors, transplants Pathologic Apoptosis Cell death produced by injurious stimuli. Radiation, anticancer drugs that damage DNA, heat, hypoxia (if mild), ER stress induced by unfolded proteins Cell injury in some viral diseases. Pathologic atrophy in parenchymal organs after duct obstruction. Cell death in tumors Can be seen with or precede oncotic necrosis Dysregulated Apoptosis (too little or too much) May be important in a wide range of diseases Disorders associated with defective apoptosis and increased cell survival Cancers. Tumors with p53 mutations common. Also hormonedependent tumors (mammary, prostate, ovary) Autoimmune disease. May arise from failure to eliminate auto reactive lymphocytes after encounter with self antigens. Disorders associated with increased apoptosis and excessive cell death. Excess loss of normal or protective cells. Neurodegenerative diseases with loss of specific neuron subsets Ischemic injury of myocardial infarcts and stroke Death of virally infected cells 7

8 Biochemical Features of Apoptosis Protein cleavage: protein hydrolysis and activation of caspase family proteases (at least 13 known). Present normally as inactive pro-enzymes that need to be activated. Results in degradation of nuclear scaffold and cytoskeleton DNA breakdown. Activation of DNAases. Breakdown to kilobase pieces with subsequent cleavage of DNA into nucleosomes in multiples of base pairs ( DNA ladders ) Phagocytic recognition. Phosphatidylserine flipped out from inner layer to outer layer permitting recognition by macrophages without inflammation. Initiation phase. Signals from 2 (or more) distinct but convergent pathways. Extrinsic (death receptor-initiated) pathway Receptor-ligand interactions: TNF receptor (TNFR1). Tumor necrosis factor family Fas Fas ligand. Active caspase 8 leads to executioner path Can be inhibited by FLIP used by some viruses to protect virally infected cells from Fas apoptosis 8

9 Extrinsic (death receptorinitiated) pathway of apoptosis 1. Fas cross linked to FasL death domains (FAD) come together and form binding site for and adaptor protein. 3. FADD attached to death receptors binds inactive caspase-8 which come together and autocatalytically activate to active caspase-8 4. Cascade of other caspase activation activates executioner caspases. 5. Apoptosis initiated Intrinsic (mitochondrial) pathway Result of increased mitochondrial permeability and release of pro-apoptotic molecules into cytoplasm (no death receptors) Initiated by many types of injury: radiation, toxins, free radicals, hypoxia, withdrawal of growth factors or hormones (which stimulate production of Bcl-2 (B cell lymphoma) family anti-apoptotic components (especially Bcl-2 and Bcl-x) that reside in mitochondrial membranes. Can be inhibited by FLIP used by some viruses to protect virally infected cells from Fas apoptosis The intrinsic (mitochondrial) pathway of apoptosis. Death agonists cause changes in the inner mitochondrial membrane, resulting in the mitochondrial permeability transition (MPT) and release of cytochrome c and other pro apoptotic proteins into the cytosol, which activate caspases. AIF, Apoptosis-inducing factor. From Kumar V, Abbas A, Fausto N: Robbins & Cotran pathologic basis of disease, ed 7, Philadelphia, 2005, Saunders.)(McGavin, M. Donald. Pathologic Basis of Veterinary Disease, 4th Edition. C.V. Mosby, 9

10 Perforin/Granzyme Pathway Cytotoxic T cells. CD8+ cells kill antigen-bearing cells. A variant of type IV hypersensitivity. A novel pathway in addition to extrinsic and FasL/FasR interaction predominantly used by CTL-induced apoptosis. Perforin, a transmembrane pore forming molecule is followed by exophytic release of cytoplasmic granules through the pore into the target cell. Granzyme A and B (serine proteases) are the major component of the granules. Perforin/Granzyme Pathway Granzyme B cleaves proteins at aspartate residues and activates pro-caspase 10 Granzyme B can also use mitochondrial path to amplify the death signal by release of cytochrome c. It can also directly activate caspase 3. Granzyme A activates caspase independent paths, activating DNA nicking via DNAase NM23-H1, a tumor suppressor gene product. Effects on virally infected cells, tumor cells, and immune modulation Execution Phase Proteolytic cascade at convergence of other paths Executioner caspase are caspase 3 and 6 Executioner caspase are caspase 3 and 6 Pro-enzymes are activated by other caspases (e.g. 8 and 9) or autocatalytically Executioner caspases: cleave cytoskeletal and nuclear matrix proteins, disrupting cytoskeleton and breaking down nucleus. 10

11 Execution Phase Nuclear targets of caspase activation; Transcription proteins DNA replication DNA repair Conversion of cytoplasmic DNAase into active form by caspase 3 cleaving of inhibitor of the enzyme. Effect is induction of internucleosomal cleavage of DNA Removal of dead cells. Phospholipid asymmetry and externalization of phosphatidylserine on the cell surface (flip) is hallmark. Surface phosphatidylserine and other molecules recruit phagocytes leading to noninflammatory phagocytic (and adjacent cell) recognition (early uptake and disposal without inflammation) Many macrophage receptors are involved in binding and engulfing apoptotic cells. Macrophages secrete substances that bind to apoptotic but not live cells resulting in opsonization for phagocytosis. Viable cells prevent engulfment by macrophages by expression of surface molecules (CD31) The result: no inflammation. Disappearance without a trace. Summary Initiators of apoptosis: TNF, nitric oxide, fas ligand, granzyme, viral infection, radiation, corticosteroids, DNA damage Inhibition of apoptosis: Growth factors, differentiation factors, adequate intracellular nutrition, insulin, others Extrinsic signaling Ligand (TNF, CD95L (Fas Ligand), Trail Activates t Pro-caspase 8t to caspase 8( (active) Intrinsic signaling DNA damage, UV damage, viral infection, cell injury -- Cytochrome c release and binds to Apaf-1 to activate caspase 9, Bcl-2 and Bcl-x (anti apoptotic) lost and replaced by Bax, Bak, etc. Initiator caspases 2, 8, 9, 10, 12 Effector caspases: 3, 6, 7 11

12 Morphology of Apoptosis Epidermal apoptosis with hypereosinophilic cytoplasm and small dense nucleus. Right. Apoptotic liver cell Apoptosis, cytoarchitecture of cells, pancreas, rat. Individual acinar cells are shrunken and their chromatin condensed and fragmented (arrows). Cytoplasmic buds are found in adjacent cells. Inflammation is absent. H&E stain. (Courtesy Dr. M.A. Wallig, College of Veterinary Medicine, University of Illinois.) (McGavin, M. Donald. Pathologic Basis of Veterinary Disease, 4th Edition. C.V. Mosby Necrosis and apoptosis, mouse hepatitis virus infection, liver, mouse. This disease causes hepatocyte death, typically by oncotic necrosis but sometimes by apoptosis. Note areas of coagulation necrosis in the lower left and apoptotic bodies in the center, some of which have been taken up by adjacent hepatocytes (arrows). H&E stain. 12

13 Assays for Apoptosis Because oncotic and apoptotic necrosis overlap, need to use 2 or more assays to confirm death by apoptosis, one for initiation phase and one for later execution phase. Understand d kinetics of cell death and realize apoptosis may be initiated and completed within 2-3 hours. False negatives can occur: too early or too late Many assays (6 major groups) are available with various strengths and weaknesses Assays for Apoptosis 1. Cytomorphologic alterations H&E visualization. Misses early stages Semi-ultrathin epoxy-resin blocks stained with Toluidine blue TEM. Gold standard to confirm apoptosis. Ultrastructural morphology. electron dense nucleus (marginalized early) nuclear fragmentation intact cell membrane, even during disintegration disorganized cytoplasmic organelles large clear vacuoles blebs (buds) on cell surface loss of cell to cell adhesions apoptotic bodies have intact cell membranes and cytoplasmic organelles, with or without nuclear fragments. Ultrastructure of apoptosis. Nuclear fragments with peripheral crescents of compact chromatin or uniformly dense fragments. 13

14 Assays for Apoptosis 2. DNA Fragmentation. 1. Laddering Technique. Endonuclease cleavage product on agarose gel. 2. TUNEL (Terminal dutp Nick End-Labeling). Assays endonuclease cleavage products by enzymatically labeling DNA strand breaks. Detection by light or fluorescence microscopy or flow cytometry. Sensitive and fast, but false positives from oncotic cells and cells in DNA repair and gene transcription. Agarose gel electrophoresis for apoptosis. A = control B = culture of heated cells showing apoptosis. Note laddering of DNA showing nucleosome-sized fragments (multiples of base pairs). C = culture of cells with massive necrosis. Note smearing of DNA Assays for Apoptosis 3. Detection of caspases, cleaved substrates, regulators, and inhibitors a. 13 known caspases detected by various caspase activity assays and immunohistochemistry. b. Caspase activation detected by western blot, immunoprecipitation, and IHC c. Apoptosis PCR microarray. Uses real time PCR to profile expression of up to 112 genes involved in apoptosis 14

15 Assays for Apoptosis 4. Membrane alterations a. Phosphatidylserine externalization on outer plasma membrane detected by Annexin V. Tissues, embryos, or cultured cells. b. Fluorescence microscopy, sensitive, but oncotic necrotic cells also labeled. c. Use dyes to mark oncotic cells Assays for Apoptosis 5. Detection of apoptosis in whole mounts 6. Mitochondrial assays a. Assays of cytochrome c release allows detection of early phase of intrinsic path b. Uses laser scanning confocal microscopy c. Can also assess mitochondrial permeability transition (MPT), calcium fluxes, mitochondrial redox, status, and reactive oxygen species d. Others including detection of apoptotic or antiapoptotic proteins (Bax, Bid, and Bcl-2) by fluorescence and confocal microscopy. Other forms of programmed cell death besides apoptosis Cell death has diverse array of phenotypes Other types of cell death may require gene activation and energy dependence. Some have features of both oncotic and apoptotic necrosis aponecrosis Autophagy may represent another mechanism of PCD Autophagic cell death has sequestration of cytoplasm and organelles in double or multimembrane vesicles and delivery to the cell s own lysosomes for degradation. Cannibalizes. Depends on protein synthesis and ATP. 15