Cell Injury and Death

Overview

Definitions

• Homeostasis: 
  • Steady state
  • Cell is at optimal function, meeting physiologic demands.
• Cellular adaptation: reversible changes in cell structures or functions in response to changes in the cell’s environment

Cell injury

• Cells cannot adapt, or the maximum adaptive response to physiologic or pathologic stimuli is exceeded.
• Occurs with damaging stimuli, loss of critical nutrients, or mutations 
• Factors affecting cell injury:
  • Nature, duration, and severity of injury
  • Type and adaptability of the cell
  • Simultaneous injury mechanisms stimulated by the etiology
• Types of cell injury:
  • Reversible injury
  • Irreversible injury (leads to cell death)
• Mechanisms of cell injury (can occur at the same time):
  • Mitochondrial damage
  • Abnormal calcium homeostasis
  • DNA damage
  • Membrane damage
  • Endoplasmic reticulum (ER) stress
  • Oxidative stress

Cell death

• State in which cell ceases to carry out functions
• Part of embryogenesis, organ development, and maintenance of homeostasis where damaged and unneeded cells are removed 
• Effect of irreversible injury, when the cell cannot overcome the damages 
• Pathways:
  • Apoptosis
  • Necrosis

Etiology and Types of Cell Injury

Injurious stimuli

• Physical agents:
  • Mechanical trauma
  • Temperature and atmospheric pressure changes
  • Radiation
  • Electric shock 
• Chemical agents and drugs:
  • Chemicals causing electrolyte derangements (e.g., glucose)
  • Poisons (e.g., cyanide, arsenic)
  • Environmental pollutants
  • Industrial hazards (e.g., asbestos)
  • Medications (toxic effects)
• Oxygen deprivation:
  • Ischemia
  • Cardiorespiratory decompensation
  • ↓ oxygen-carrying capacity of the blood (anemia)
• Infections: viruses, bacteria, parasites, other biological agents
• Immunologic reactions:
  • Immune reactions to external agents
  • Autoimmune diseases 
• Genetic abnormalities:
  • Congenital malformations
  • Deficient protein function from enzyme defects
  • Misfolded proteins 
• Nutritional deficiencies and excess:
  • Nutritional deficiencies (vitamin deficiency)
  • Nutritional excess (obesity, increased lipids)

Types of cell injury

• Reversible cell injury:
  • Effect of mild or transient damage 
  • Elimination of the pathologic stimuli or restoration of critical supply leads to cell returning to steady state.
  • Features:
    • Cellular swelling due to water influx (earliest manifestation of cell injury)
    • Hydropic change or vacuolar degeneration: small, clear vacuoles within the cytoplasm (from distended ER)
    • Plasma membrane alterations (blebbing, blunting, loss of microvilli)
    • Mitochondrial swelling and appearance of amorphous densities
    • ↑ cytosol myelin figures (phospholipids from damaged membranes)
    • Changes in nucleus (granular and fibrillar elements disaggregate) 
    • Fatty change from accumulation of lipid vacuoles (in organs involved in lipid metabolism)
• Irreversible injury:
  • “Point of no return”: cell cannot be restored → cell death 
  • Injurious stimulus is severe and/or persistent.
  • Features:
    • Inability to reverse mitochondrial dysfunction (loss of oxidative phosphorylation and adenosine triphosphate (ATP) production)
    • Significant damage to membrane function

Cell Injury by Mitochondrial Damage

Mitochandria

• Major site of synthesis of adenosine triphosphate or ATP 
• ATP: 
  • Energy required in synthetic and degradative processes
  • Sources:
    • From oxidative phosphorylation of adenosine diphosphate 
    • From the glycolytic pathway (anaerobic)
• Subject to most injurious stimuli

Consequences of mitochondrial damage

• Adenosine triphosphate (ATP) depletion: mitochondrial permeability transition pore forms → loss of membrane potential and oxidative phosphorylation → reduced ATP
• ATP depletion leads to:
  • Cell swelling: plasma membrane sodium pump (Na⁺, K⁺-ATPase) fails → sodium enters the cell → water accumulation
  • Reduced cytosolic enzyme activity: ↑ glycogenolysis and glycolysis to compensate for the ATP loss → depleted glycogen → ↑ lactic acid and inorganic phosphates → ↓ intracellular pH → impaired enzymes
  • Reduced protein synthesis: detachment of ribosomes 
• Impaired oxidative phosphorylation: Reactive oxygen species (free radicals) are created, causing lipid/protein/nucleic acid damage.
• Apoptosis/cell death: leakage of apoptotic proteins (e.g., cytochrome c) results in organelle damage

Cell Injury by Abnormal Calcium Homeostasis

Calcium homeostasis

• Intracellular calcium (Ca²⁺): normally low (sequestered in the mitochondria and ER)
• Common injurious stimuli: 
  • Oxygen deprivation/ischemia
  • Toxins 

Consequences of impaired calcium homeostasis

• Release of Ca²⁺ from intracellular stores and ↑ Ca²⁺ influx through the plasma membrane 
• Increased Ca²⁺ activates enzymes, which produce cell injury: 
  • Phospholipases → membrane damage
  • Proteases → membrane and cytoskeletal protein degradation
  • Endonucleases → DNA and chromatin fragmentation
  • ATPases → ATP depletion

Cell Injury by DNA and Membrane Damage

DNA damage

• Common injurious stimuli: 
  • Radiation
  • Chemotherapeutic drugs
  • ROS
• May be part of aging 

Consequences of damaged DNA

• Triggers p53 pathway: arrests cell cycle in G1 phase, activating repair mechanisms
• Apoptosis occurs:
  • If repairs cannot correct the damage 
  • To protect the tissue involved (cell dies rather than persists with an abnormal DNA, which has potential for malignant transformation)

Membrane damage

• Normal membrane: made of lipids, with phospholipids as the most abundant form
• Common injurious stimuli: 
  • Bacterial infection (toxins)
  • Viral proteins
  • Complement-mediated lysis
  • Physical/chemical agents 
  • Ischemia
• Other mechanisms overlap and cause membrane damage:
  • Oxygen free radicals → lipid peroxidation → phospholipid loss
  • Mitochondrial damage → reduced ATP → decreased phospholipid synthesis
  • Calcium-dependent phospholipases → phospholipid breakdown → loss of membrane
  • Calcium-dependent proteases → cytoskeletal filament damage → increased cellular swelling and rupture 

Consequences of membrane damage

• ↑ permeability of plasma membrane → influx of fluids and ions + loss of cell osmotic balance
• Injury to lysosomal membranes → lysosomal enzymes disrupt cytoplasmic organelles

Cell Injury by ER Stress

Endoplasmic reticulum

• Site of protein synthesis and folding, lipid synthesis, and free calcium storage
• Chaperones: control protein folding 
• Misfolded proteins: usually processed for proteolysis
• Unfolded protein response: 
  • Signal transduction pathways that sense misfolded proteins
  • ↑ chaperones, ↓ protein translation, and ↑ degradation of misfolded proteins
• Injurious stimuli: 
  • Genetic abnormalities/mutations
  • Ischemia/hypoxia 
  • Viral infections

Consequences of ER stress

• ER stress: Protein folding demand exceeds protein folding capacity.
• Unrepaired proteins accumulate → apoptosis
• Diseases and their associated misfolded proteins:
  • Cystic fibrosis: cystic fibrosis transmembrane conductance regulator (CFTR)
  • α1- antitrypsin deficiency: α1- antitrypsin
  • Alzheimer’s disease: Aβ peptide
  • Familial hypercholesterolemia: LDL receptor
  • Creutzfeldt-Jacob disease: prion

Cell Injury by Oxidative Stress

Free radicals

• Molecular species with single unpaired electron in the outer orbit
• Highly reactive: attack adjacent molecules (proteins, carbohydrates, nucleic acids)
• ROS: an oxygen-derived free radical
• Principal free radicals:
  • Superoxide anion (O₂^–)
  • Hydrogen peroxide (H₂O₂)
  • Hydroxyl radical (^•OH): most reactive ROS
  • Peroxynitrite (ONOO⁻)
• Injurious stimuli: 
  • Ischemia-reperfusion injury
  • Chemical and radiation injury
  • Aging
  • Phagocytosis of microbes

Oxidative stress

• Accumulation of ROS → oxidative stress:
  • From increased production of free radicals
  • From decreased scavenging of ROS
• The following generate free radicals:
  • Reduction-oxidation reactions:
    • O₂ is reduced with transfer of electrons to H₂ to form water molecules.
    • Partially reduced intermediates → free radicals
  • Exposure to ionizing radiation and ultraviolet rays
  • Polymorphonuclear neutrophils produce free radicals during inflammatory response.
  • Metabolism of exogenous chemicals (e.g., carbon tetrachloride)
  • Reactions with transition metals (e.g., iron or copper)
  • Nitric oxide reaction (with superoxide) in macrophages, producing peroxynitrite (ONOO⁻), a free radical

Consequences of oxidative stress

• Membrane damage by lipid peroxidation:
  • ROS attack unsaturated fatty acids of the membrane.
  • Lipid hydroperoxides are produced → ↓ function of membranes
• DNA damage or fragmentation 
• Oxidative modification of proteins: ↑ protein cross-linking leads to ↑ degradation and ↓ activity

Antioxidants

• Defense against free radicals (ROS scavengers)
• Immediately eliminate ROS produced during mitochondrial respiration and energy generation
• Low amount of free radicals may be present but are unable to induce damage.
• Non-enzymatic mechanism:
  • Vitamins A, C, E
  • Glutathione
  • Ferritin
  • Transferrin
  • Ceruloplasmin
• Enzymatic mechanism:
  • Glutathione peroxidase: catalyzes breakdown of hydroxyl radicals
  • Superoxide dismutase (SOD): converts superoxide to hydrogen peroxide (H₂O₂)
  • Catalase: breaks down H₂O₂

Cell Death by Apoptosis

Programmed cell death (apoptosis)

• Activated enzymes degrade DNA and proteins in cells that are destined to die
• Features:
  • ↓ cell size, eosinophilic cytoplasm
  • Chromatin condensation (chromatin aggregates peripherally)
  • Cytoplasmic blebs and apoptotic bodies
  • Phagocytosis of apoptotic cells by macrophages

Apoptosis in different conditions

• Physiologic conditions:
  • Fetal development: 
    • Cells die off after their purpose has been fulfilled.
    • Removal of supernumerary cells during development
  • Involution of tissues with hormone withdrawal:
    • Endometrial shedding in menstrual cycle
    • Lactating breast regression (weaning)
  • Removal of self-reactive lymphocytes (may cause autoimmune disease)
  • Removal of neutrophils in an inflammatory response
  • Control of cell proliferation, maintaining a constant number of cell populations (immature lymphocytes in bone marrow) 
• Pathologic conditions:
  • DNA damage: Apoptosis prevents survival of cells with DNA mutations (protective effect).
  • Removal of improperly folded proteins
  • Ductal obstruction (e.g., kidney, parotid gland): Atrophy occurs by apoptosis. 
  • Infections (particularly viral illness): Cytotoxic T lymphocytes induce apoptosis to eliminate infected cells.

Mechanisms of Apoptosis

Caspases:

• Cystein aspartic acid proteases
• Exist in inactive form, requiring enzymatic cleavage to be activated
• Active caspases: a marker for cells undergoing apoptosis
• Phases of apoptosis:
  • Initiation: activation of caspases → cascade of other caspases
    • Intrinsic pathway
    • Extrinsic pathway
  • Execution: terminal caspases → cellular fragmentation

Intrinsic pathway (initiation):

• Mitochondrial pathway
• In viable cells, growth factors and survival signals reduce mitochondrial leakage of cytochrome c by producing anti-apoptotic proteins (principal members):
  • BCL2
  • BCL-XL
  • MCL-1
• In damaged cells, loss of survival signals, DNA damage, protein misfolding: 
  • Allow cytochrome c leakage from the mitochondria by producing pro-apoptotic proteins (main members):
    • BAX
    • BAK 
  • Activate apoptosis initiators (BH3-only proteins): BAD, BIM, BID, Puma, Noxa
• Events:
  • Increased permeability of the mitochondrial outer membrane → release of cytochrome c into the cytoplasm 
  • Cytochrome c initiates apoptosis.
  • In the cytoplasm, cytochrome c binds with apoptosis-activating factor-1 (APAF-1), forming a structure, apoptosome.
  • Apoptosome leads to self-cleavage and activation of caspase-9, the initiator caspase.
  • Activated caspase-9 → cascade of executioner caspases 

Extrinsic pathway (initiation):

• Death receptor-initiated pathway
• Plasma membrane death receptors initiate this pathway.
• Death receptors: 
  • Members of tumor necrosis factor (TNF) family with a cytoplasmic death domain (delivers the apoptotic signals)
  • Best known death receptors: 
    • Type 1 TNF receptor (TNFR1)
    • Fas (CD95)
• Events:
  • FasL (Fas ligand on T cells and cytotoxic T lymphocytes) binds to Fas → a signal for apoptosis is given to the cell.
  • 3 or more Fas molecules combine to form the protein, Fas-associated death domain (FADD).
  • FADD binds pro-caspase-8.
  • Caspase-8 (or caspase-10) is activated → stimulates executioner caspases 

Execution phase:

• Both pathways converge in the execution phase.
• Events:
  • Starts with sequential activation of executioner caspases.
  • Inhibitor of deoxyribonuclease (DNase) is cleaved → active DNase → nuclear proteolysis and fragmentation
  • Cytoskeleton proteins break down.
  • Cell fragments → cytoplasmic blebs form → become apoptotic bodies
  • Apoptotic bodies are eaten by phagocytes.
• Efferocytosis: 
  • Apoptotic cell phagocytosis
  • Rapid clearance with reduced production of pro-inflammatory cytokines → limited inflammatory reactions even with substantial apoptosis

Clinical correlation

• Tumor suppressor genes: 
  • Prevent uncontrolled cell proliferation and lead cells to apoptosis. 
  • Inactivation → malignant neoplasm
• Follicular lymphoma:
  • Associated with chromosomal translocations involving BCL2 gene
  • Has BCL2 over-expression
  • Increased anti-apoptotic mechanisms → expansion of malignant cells

Cell Death by Necrosis

Process of necrosis

• Uncontrolled cell death after irreversible injury:
  • Cell membrane is disrupted; lysosomal enzymes enter and digest the cell.
  • Cellular contents are released and circulate into the extracellular space. 
  • Circulating contents elicit an inflammatory reaction and recruit leukocytes to the site of necrosis.
• Clinical correlation (tests for tissue-specific injury represent circulating intracellular contents):
  • Troponin: from damaged cardiac muscle cells
  • Alkaline phosphatase: from bile duct epithelium
  • Transaminases: from hepatocytes

Cellular changes

• Cytoplasmic changes: 
  • Eosinophilic cytoplasm: due to denatured cytoplasmic proteins (which bind to eosin dye) 
  • Vacuolated cytoplasm: Enzymes digest organelles, leaving “moth-eaten” appearance.
  • Myelin figures: large whorled phospholipid precipitates (from the damaged membrane), which are phagocytosed or degraded to fatty acids
• Nuclear changes (1 of 3 patterns):
  • Karyolysis: reduced basophilia due to DNA loss (effect of DNAse)
  • Pyknosis: nuclear shrinkage and increased basophilia (condensation of chromatin into a dense basophilic mass)
  • Karyorrhexis: fragmentation of the nucleus

Patterns of Necrosis

Coagulative necrosis:

• Cell outlines and tissue architecture maintained for several days
• Injury also denatures enzymes, so initial proteolysis is blocked.
• Eventually, leukocyte enzymes break down the dead cells.
• Often in ischemia or hypoxic injury
• Infarct: localized area of coagulative necrosis 
• Seen in myocardial and renal infarction

Liquefactive necrosis:

• Colliquative necrosis
• Tissue is digested and dissolved into a viscous liquid.
• Seen in bacterial and fungal infections, which stimulate leukocytes and the release of hydrolytic enzymes
• Pus: creamy-yellow necrotic material 
• Default necrosis mechanism used by hypoxic central nervous system cells
• Coagulative and liquefactive necrosis: not mutually exclusive
• Damaged cardiac myocytes undergo coagulative necrosis; as leukocytes set in and enzymes are released, liquefactive necrosis occurs.

Caseous necrosis :

• Caseous: “cheese-like”
• Fragmented cells and debris surrounded by an inflammatory border: granuloma
• Seen in tuberculosis and some fungal infections
• Mycolic acid from the mycobacterial cell wall induces granuloma formation.

Fat necrosis:

• Change in adipose tissue due to trauma or enzymatic release
• Release of pancreatic lipases into the pancreatic parenchyma and the peritoneum → destruction of adipocytes 
• Liberated fatty acids combine with calcium, producing chalky-white areas (fat saponification).
• Seen in acute pancreatitis, fat necrosis of the breast

Fibrinoid necrosis:

• Microscopic change
• Deposition of immune complexes in the walls of vessels
• Fibrinoid: fibrin combined with immune complexes deposited in the vessel walls (homogeneously pink in hematoxylin and eosin stains)

Gangrenous necrosis:

• Not a pattern of necrosis; a clinical description used when a limb becomes necrotic due to ischemia 
• Indicates coagulative necrosis of multiple layers of tissue (dry gangrene)
• With superimposed bacterial infection, liquefaction necrosis occurs due to enzymes from bacteria and leukocytes (wet gangrene).

Dystrophic calcification:

• Necrotic cells: eliminated by enzymatic digestion and phagocytosis
• Inadequately reabsorbed necrotic cells become a nidus of calcium and mineral deposition.

Other Cellular Mechanisms

Necroptossis

• Programmed necrosis
• Caspase-independent cell death
• Similar to necrosis in morphology, with apoptotic type of cell death 
• Clinical correlation:
  • Physiologic: mammalian growth plate
  • Pathologic: steatohepatitis, Parkinson’s disease

Pyroptosis

• Apoptosis accompanied by cytokine IL-1 (interleukin-1: fever-inducing cytokine)
• Pathway of apoptosis releases inflammatory mediators.
• Clinical correlation: death of cells infected by microbes 

Ferroptosis

• Iron-dependent pathway of cell death, characterized by lipid peroxidation
• Results in the loss of membrane permeability (ruptured mitochondrial membrane)
• Occurs with excessive iron or ROS, which glutathione-dependent defenses cannot handle
• Clinical correlation: cancer, neurodegenerative diseases, and stroke

Autophagy

• “Auto”: self; “phagy”: eat → cell eats its contents
• Survival mechanism, such as in atrophic cells in states of nutrient deprivation
• Triggers cell death, if unable to cope with stress
• Mechanism:
  • Nucleation and formation of a phagophore, an isolation membrane derived from the ER or mitochondria or plasma membrane
  • The organelles are sequestered by the phagophore and a vesicle, autophagosome, forms.
  • Mature autophagosome fuses with a lysosome (autophagolysosome), resulting in the degradation of contents.
• Clinical correlation:
  • Cancer
  • Neurodegenerative disorders (e.g., Alzheimer’s disease)
  • Infectious disease
  • Inflammatory bowel disease