LMP 300Y Lecture 1 Cell injury, adaptation & death Douglas M. Templeton, Ph.D., M.D. doug.templeton@utoronto.

ca
References Kumar, Cotran & Robbins, Basic Pathology, 7th ed.Saunders, 2003. Apoptosis - Nature, Oct. 12, 2000; Annu. Rev. Pharmacol. Toxicol., 42:259 (2002) Some images from http://medlib.med.utah.edu/WebPath/webpath.html
2002/2003; revised 2005

Cell death -- good and bad - good: development, T cell clones, cancer cells - bad: tissue destruction, atrophy Four terms: Necrosis Apoptosis Necrapoptosis Anoikis

CAUSES OF CELL INJURY:

Internal stresses metabolic imbalances, nutritional deficiencies or excesses genetic abnormalities acquired derangements > hypoxia, ischemia External physical agents (heat, cold, radiation, ) natural toxins, venoms drugs, "chemicals" (Paracelsus)

RESPONSES TO INJURY:
Recovery Adaptation Death Depends on mechanism, severity, duration,

Cell injury may be reversible or irreversible

Stages in Cell Injury

Cellular function is lost far before cell death occurs, and the morphologic changes of cell injury (or death) lag far behind both.

"Even at the level of the light microscope, it is apparent that cells exhibit a finite number of morphologic reactions to a wide range of internal and external environmental stresses." "This implies common biochemical and molecular mechanisms responsible for cell adaptation and failure of adaptation, or cell death."

Different cells show different sensitivities/thresholds. Exam ples:

Brain cells, heart cells susceptible to hypoxia and ischemia; liver cells susceptible to chem ical injury . Calf muscle tolerates 2-3 h of ischemia, cardiac m uscle dies in 20-30 min. Highly differentiated surface epithelial cells of the respiratory tract more susceptible to cigarette smoke than less differentiated basal epithelia. Nutritional status glycogen-replete hepatocyte more resistant to ischem ia than depleted one.

 Hypoxia - Oxygen deficiency Ischemia - Impaired blood supply

(arterial or venous occlusion)

Infarction - Area of necrosis due to ischemia

Some basic types of tissues

Epithelium, endothelium Connective tissue, fibroblasts Muscle tissue smooth, skeletal, cardiac Nervous tissue Blood and lymph

A Classification of Epithelium
Simple
Simple squamous (endothelium) Simple cuboidal (renal tubule) Simple columnar (small intestine)

Stratified squamous
Low keratin (esophagus) Keratinized (epidermis)

Pseudostratified
Columnar, ciliated (trachea, epididymis) Transitional (bladder)

FOUR VULNERABLE SYSTEMS: Cell membrane integrity ATP generation / mitochondrial function Protein synthesis / enzyme function Genetic integrity

SIX GENERAL MECHANISMS: ATP depletion (ox/phos or glycolysis) Oxygen (i) ischemia/hypoxia Oxygen (ii) ROS Loss of Ca2+ homeostasis Plasma membrane integrity Mitochondrial damage

ATP

O2

ROS

Ca2+ PM

Mito. fcn.

A CENTRAL ROLE OF FREE RADICALS IN CELL DEATH

Sources Mitochondrial respiration Xanthine oxidase (purine metabolism > uric acid, O2-.) Peroxisomes (long chain FA > H2O 2) NADPH oxidase (respiratory burst) Cyt P450 mixed function oxidase

Defences Glutathione Catalase (H2O 2) - peroxisomes Mn-superoxide dismutase - mitochondria Cu,Zn-SOD - cytosol Antioxidants Metal sequestration Metallothionein

QuickTime and a Photo - JPEG decompressor are needed to see this picture.

ADAPTIVE RESPONSES OF CELLS: Atrophy Hypertrophy Hyperplasia Metaplasia Storage

ATROPHY: Cell shrinkage by loss of substance

Cerebral atrophy - Alzheimer disease

Testicular Atrophy

HYPERTROPHY: Increase in cell (hence organ) size

QuickTime and a Photo - JPEG decompressor are needed to see this picture.

Hypertrophy - normal and gravid uterus

QuickTime and a Photo - JPEG decompressor are needed to see this picture.

QuickTime and a Photo - JPEG decompressor are needed to see this picture.

HYPERPLASIA: Increase in cell number

METAPLASIA: (Reversible) replacement of one differentiated cell type by another

QuickTime and a Photo - JPEG decompressor are needed to see this picture.

STORAGE:

Normal Liver

Fatty Liver

Fatty Liver

Hemochromatosis

Calcification - Tricuspid valve

OVERVIEW
i) Atrophy decreased testosterone > prostatic atrophy (apoptosis) ii) Hypertrophy exercise / skeletal muscle; hypertension / cardiac myocyte iii) Hyperplasia hyperthyroidism, effect of excess TSH on thyroid gland iv) Metaplasia ciliated epithelium > squamous epithelium in smoker. (Point for argument: Is the myofibroblast a metaplastic cell?) v) Storage Gaucher's disease (glucocerebrosidase),Haemochromatosis (Fe), Fatty liver (EtOH)

Some terms in the histology of cell injury:

Fluid or fat accumulates in vacuoles cloudy swelling / hydropic degeneration e.g., disruption of ion transport/pumping (loss of ATP > Na+/K+ ATPase, oxidation of thiols on pumps, disorganization of membrane lipids, ) Fat accumulation fatty change - fatty acid synthesizing/transporting cells (heart, liver, kidney) - ER membrane damage, FA oxid'n, TG synth., lipoprotein synth., Irreversible injury: A cell may be irreversibly injured long before any changes are apparent in the microscope. Coagulation necrosis influx of water and ions, mitochondrial swelling, general loss of membrane integrity, influx of Ca2+ (coagulation of proteins, activation of enzymes), release of lysosomal enzymes ( autolysis)

Kidney Infarct - coagulative necrosis

Cerebral Infarct - liquefactive necrosis

Caseous necrosis - tuberculosis

APOPTOSIS
Membrane blebbing, cell shrinkage, protein fragmentation, chromatin condensation, DNA degradation, engulfment - central role of caspases, cysteine proteases cleaving Asp-Xxx bond - upstream (initiator) and downstream (effector) caspases - may inactivate (e.g., lamins) or activate (e.g., nucleosomal nuclease) substrate

Apoptosis vs. Coagulation Necrosis Apoptosis Stimulus Physiological (Developmental, Atrophy, ) Selected Pathological Single cells, shrinkage, chromatin condensation, apoptotic bodies Intact Chromatin condensation, internucleosomal breaks, laddering (karyorrhexis) Phagocytosis of apoptotic bodies Necrosis Hyppoxia, Toxins

Histology Organelles Nucleus

Cell swelling, groups of cells, tissue disruption Swelling of mitochondria & ER Disappearance, Random DNA breaks (karyolysis) Inflammation, regeneration or repair by fibrosis

Outcome

Extrinsic

Intrinsic

Bcl-2 family members balance between pro-apoptotic (e.g., Bax, Bak) and anti (e.g., Bcl-2, Bcl-x) determines outcome. Hydrophobic C-terminal domain localizes them to outer mitochondrial membrane. With other proteins, form channels to facilitate release of Cyt c. Mitochondrial permeability transition pore MPTP

Caspases are synthesized as inactive zymogen; pro-domain, p20,

and p10 domains. Activated by cleavage between p20 and p10, and prodomain and p20. Active as tetramer of 2 p10 and 2 p20 domains. Three models for caspase activation. i) caspase cascade, e.g. downstream effectors caspase-3, -6, -7 ii) induced proximity, e.g., on ligand binding CD95 receptors aggregate to form signaling complexes, which through adapter proteins bring about high local concentrations of procaspase-8 iii) association with a regulatory subunit, e.g., caspase-9 and Apaf-1

DNA damage can initiate apoptosis. Dual function of p53: If damage detected, cell cycle arrest. If damage not repaired, iniates apoptosis. How is damage sensed? Proteins of the ATM (ataxia telangiectasiamutated) and DNA-PK contain DNA binding domains and protein kinase activity. Both phosphorylate p53.

Signals for ingestion: i) altered sugars recognized by lectins on macrophages ii) Thrombospondin secreted by macrophages, binds to apoptotic cells (mechanism not known), then macrophage integrins bind to thrombospondin. iii) phosphatidyl serine (annexin V)

Apoptosis can be suppressed at the level of caspases at the level of the mitochondria by ionic control

QuickTime and a GIF decompressor are needed to see this picture.

Necrapoptosis Lemasters, Am. J. Physiol. 276: G1-G6 (1999). Cell balanced between apoptosis and necrosis depending on production of ATP. Anoikis Frisch & Ruoslahti, Current Opin. Cell Biol. 9: 701-706 (1997). "Homelessness". Apoptosis initiated by detachment of epithelial cell from matrix.