Pathology 3: Irreversible cell death - Apoptosis

What happens when there is too little apoptosis?

Defective apoptosis and increased cell survival: - neoplasia - autoimmune disorders

How does DNA damage-mediated apoptosis occur?

Exposure of cells to radiation or chemotherapeutic agents induces apoptosis - p53 accumulates when DNA is damaged and arrests the cell cycle --> if DNA repair process fails, p53 induces pro-apoptotic members of BCl family - leakage of cytochrome c - activation of caspases

What happens when there is too much apoptosis?

Increased apoptosis and excessive cell death - death of virus-infected cells (ex. Feline panleukopenia) - autoimmune disorders (EM)

Which caspases are associated with the initiation phase versus the execution phase of apoptosis?

Initiation phase: caspase-8 and caspase-9 Execution phase: caspase-3 and caspase-6

How does mitochondrial damage act as a mechanism for cell injury?

Leads to decreased ATP production and may start the intrinsic pathway of apoptosis

What is ischemia?

Loss of blood supply from impeded arterial flow or reduced venous drainage in a tissue - compromises the delivery of substrates for glycolysis

What is hypoxia?

Partial reduction in the oxygen concentration supplied to cells or tissue - causes cell injury by reducing oxidative respiration - one of the most common and important causes of cell injury and death - glycolytic energy production can continue

What is the extrinsic pathway of apoptosis?

- initiated by engagement of cell surface death receptors (outside cell) Ex. Fas protein on virally infected cell binds its ligand - Fas-L on T lymphocyte and initiates activation of caspase-8. --> triggers execution of apoptosis

What is the intrinsic (mitochondrial) pathway of apoptosis?

- result of increased mitochondrial permeability and release of pro-apoptotic molecules into the cytoplasm, without the involvement of death receptors - growth factors usually keep cell in anti-apoptotic state - if cell is deprived of survival signals or is stressed, pro-apoptotic signals are induced (Bak, Bax) --> leakage of cytochrome C into cytosol, where it binds to Apaf-I and triggers activation of caspase-9 which triggers execution of apoptosis

What are 7 mechanisms of cell injury?

1. ATP depletion 2. Mitochondrial damage 3. Loss of calcium homeostasis 4. Oxygen-derived free radicals (oxidative stress) 5. Free radical generation 6. Cellular defences against free radicals 7. Defects in membrane permeability

What are three defenses against free radicals?

1. Antioxidants - Vitamin E, A, and C - glutathione - block the initiation of free radical formation or inactivate them 2. Metal binding proteins - iron and copper can catalyze the formation of reactive oxygen species - the levels of these reactive forms are minimized by iron and copper binding proteins (transferrin, lactoferrin, ceruloplasmin) 3. Enzymes acting as free radical-scavenging systems - superoxide dismutase converts superoxide to H2O2 - catalase is present in peroxisomes which decompose H2O2

What are three examples in which apoptosis occurs?

1. Apoptosis after growth factor deprivation 2. DNA damage-mediated apoptosis 3. Cytotoxic T-lymphocyte-mediated apoptosis

Give five examples of pathologic conditions which use apoptosis

1. Cell death of infected or neoplasticism cells induced by cytotoxic T cells 2. DNA damaged by radiation and cytotoxic anticancer drugs 3. ER stress induced by the accumulation of unfolded proteins 4. Pathologic atrophy in parenchyma organs after duct obstruction (hydronephrosis) 5. Cell death in tumors

What are six characteristic morphologic changes associated with apoptosis?

1. Cell shrinkage 2. Chromatin condensation* and nuclear fragmentation 3. Formation of cytoplasmic bless and apoptotic bodies 4. Phagocytosis of apoptotic cells or cell bodies 5. Intact plasma membranes 6. No inflammation

What are the 8 steps of ischemic cell injury?

1. Decrease of oxidative phosphorylation and ATP 2. Increased glycolysis, increased intracellular lactate, and depletion of glycogen stores 3. Failure of Na-K pump due to ATP deficiency 4. Net influx of Na, Ca, and H2O with loss of intracellular K and Mg 5. Swelling of mitochondria and cytocavitary network 6. Detachment of ribosomes, clumping of nuclear chromatin, loss of microvilli, vesiculation of ER, formation of membrane whorls 7. Severe disruption of cell membranes and swelling of mitochondria, influx of calcium into mitochondria and cytosol, intracellular release of lysosomal enzymes, overall cell enlargement, and clearing of the cytosol 8. Leakage of cellular enzymes, cell death (necrosis), phagocytosis

Give three examples of physiologic apoptosis

1. Embryogenesis (implantation, organogenesis, developmental involution, metamorphosis) 2. Hormone-dependent involution in the adult (endometrial cell breakdown and regression of mammary glands) 3. Lymphocytes at the end of an immune response

What are 6 important characteristics of apoptosis?

1. Energy dependent 2. Shrinkage and fragmentation 3. Plasma membrane remains intact 4. Targeted and rapidly cleared by phagocytes 5. No leakage of cellular contents 6. No inflammatory reaction

What are the two pathways/stimuli involved in the initiation phase of apoptosis?

1. Extrinsic (receptor-initiated) pathway 2. Intrinsic (mitochondrial) pathway

What are 8 possible causes of cell injury?

1. Hypoxia 2. Ischemic 3. Physical agents 4. Infectious agents 5. Immunological reactions 6. Genetic derangement 7. Nutritional imbalances 8. Chemical agents

Give four examples of hypoxia

1. Inadequate oxygenation of blood due to cardio respiratory failure 2. Anemia (decreased number of RBCs) 3. Carbon monoxide poisoning 4. Blockage of cell respiratory enzymes (cyanide toxicosis)

Give three examples of ischemia

1. Infarction 2. Heart failure 3. Shock

What are the two phases of apoptosis?

1. Initiation phase - caspases become catalytically active 2. Execution phase - Enzymes act to cause cell death

What three ways does oxidative stress act as a mechanism for cell injury?

1. Lipid peroxidation of plasma and organelle membranes 2. Oxidative modification of proteins 3. DNA damage

Give four examples of how physical agents can cause cell injury

1. Mechanical trauma 2. Extremes of temperature 3. Radiation 4. Electric shock

What types of defects in membrane permeability can occur?

1. Mitochondrial dysfunction 2. Loss of membrane phospholipids 3. Cytoskeletal abnormalities 4. Reactive oxygen species 5. Accumulation of lipid breakdown products 6. Early loss of selective membrane permeability leading to overt membrane damage is a consistent feature of most forms of cell injury 7. Injury to lysosomal membranes results in leakage of their enzymes into the cytoplasm and activation of these enzymes

What are four ways that reperfusion injury occurs?

1. New damage initiated by increased generation of oxygen free radicals from affected tissue and infiltrated neutrophils 2. Restored blood flow results in tremendous influence of calcium which enters cells 3. Ischemic injury associated with inflammation -> neutrophils recruited and inflammation causes additional injury 4. Activation of complement pathway - some IgM Abs are deposited in ischemic tissues and when blood flow resumes, complement binds the Abs, are activated, and cause cell injury and inflammation

Give four examples of infectious agents that cause cell injury

1. Viruses 2. Bacteria 3. Fungi 4. Protozoa and meatloaf parasites

How does CTL-mediated apoptosis occur?

CTLs recognize foreign antigens presented on the surface of infected host cells - CTLs secrete performing which allows entry of the CTL granzyme B --> cleaves proteins at aspartate residues and activates cellular caspases

What is the execution phase?

Caspase-3 and -6 cleave cytoskeletal and nuclear matrix proteins and activate endonucleases with ultimate cellular fragmentation and formation of apoptotic bodies

How does loss of calcium homeostasis act as a mechanism for cell injury?

Cytosolic calcium usually maintained at extremely low concentrations (<0.1micromolar) Increased cytosolic calcium can cause activation of various enzymes that have deleterious effects: - ATPases - phospholipases - proteases - endonucleases Increased intracellular calcium may also result in increased mitochondrial membrane permeability and induction of apoptosis

How does apoptosis after growth factor deprivation occur?

Hormone sensitive cells deprived of hormones are triggered to undergo apoptosis by the intrinsic pathway - excess of pro-apoptotic members of Bcl family

How are free radicals generated?

Iron and copper donate or accept free electrons during intracellular reactions and catalyze free radical formation Nitric oxide, an important chemical mediator generated by endothelium and macrophages can act as a free radical Radiant energy can hydrolysis water into hydroxyl and hydrogen free radicals Enzymatic metabolism of exogenous chemicals or drugs

Are tissues injured more rapidly and severely in ischemic or hypoxic tissues?

Ischemic tissues - anaerobic energy generation stops after glycolytic substrates are exhausted or glycolytic function becomes inhibited by the accumulation of metabolites that would have been otherwise removed by blood

What is apoptosis?

Pathway of cell death that is induced by a tightly regulated intracellular energy dependent program in which cells destined to die activate enzymes that degrade the cells' own nuclear DNA and nuclear and cytoplasmic proteins

What is reperfusion injury?

Restoration of blood flow to ischemic tissues can result in recovery of cells if they are reversibly injured, BUT, if irreversible cell damage has occurred, reperfusion will not affect the outcome - New damaging processes are set in motion causing the death of cells that might have recovered otherwise

Why is there no inflammatory reaction associated with apoptosis?

The dead cell is rapidly cleared before its contents have leaked out

What causes white muscle disease?

Vitamin E/Se deficiency - mediated by oxygen free radicals