Pathology Ch2 - Cellular Responses to Stress and Toxic Insults - pp31-68

Overview: Cellular Responses to Stress and Noxious Stimuli

Adaptations = reversible functional/structural responses to changes in phsyiologic states that allow the cell to survive & fxn
o Ex. hypertrophy, hyperplasia, atrophy, or metaplasia
o Reversible if stress is eliminated
Injurious agents/stress or limited adaptive response lead to cell injury
o Reversible up to a certain point
o Irreversible if stimulus persists or is severe > cell death (necrosis or apoptosis)
Nutrient deprivation > autophagy
Metabolic derangements > intracellular accumulations
Cell death > pathologic calcification
Cellular aging > characteristic morphologic and functional changes

Adaptations of Cellular Growth and Differentiation

Hypertrophy
o Increase in size of cells due to synthesis of additional intracellular structures > increase size of affected organ
o Physiologic = due to inc. functional demand or stimulation by hormones/growth factors
o Pathologic = ex. heart hypertrophy
o Mechanisms of Hypertrophy
Ex. heart hypertrophy
Mechanical sensors, growth factors, and vasoactive agents > signal transduction pathways
PI3K/AKT pathway > G-protein-coupled receptors > GATA4, NFAT, MEF2 transcription factors
Result of increased production of cellular proteins
Reaches a limit beyond which enlargement of muscle mass is not longer able to cope w/ inc. burden
Hypertrophy of the heart becomes maladaptive > lysis and loss of contractile elements > heart failure,
arrhythmias, sudden death

Hyperplasia
o Increase in number of cells in response to a stimulus
o Frequently occurs w/ hypertrophy
o Physiologic Hyperplasia
Due to action of hormones or growth factors
When there is a need for inc. functional capacity of hormone sensitive organs (ex. breast @ pregnancy)
When there is a need for compensatory increase after damage/resection (ex. liver)
o Pathologic Hyperplasia
Caused by excessive or inappropriate action of hormones or growth factors
Ex. endometrial hyperplasia, benign prostatic hyperplasia
Hyperplasia regresses if hormonal stimulation is elminated, but can provide a backdrop for cancer
o Mechanisms of Hyperplasia
Result of growth-factor-driven proliferation of mature cells, or inc. output of new cells from stem cells

Atrophy
o Reduction in size of an organ or tissue due to decrease in cell size and number
o Physiologic atrophy common during normal development (ex. embryonic structures)
o Pathologic atrophy
Decreased workload (atrophy of disuse)
Loss of innervation (denervation atrophy)
Diminished blood supply (ex. senile atrophy of the brain and heart)
Inadequate nutrition (ex. protein-calorie malnutrition "marasmus" > muscle wasting "cachexia")
Loss of endocrine stimulation (ex. postmenopausal estrogen loss)
Pressure/tissue compression (likely result of ischemic changes due to compression of vasculature)
o Mechanisms of Atrophy
Results from decreased protein synthesis and inc. protein degradation in cells
Degradation of cellular proteins via ubiquitin-proteasome (activated by nutrient deficiency or disuse)
Can be accompanied by inc. autophagy (starved cell eats its own components to reduce nutrient demand)
 Metaplasia
o Reversible change in which one differentiated cell type is replaced by another cell type
o Persistent stimulus and metaplasia > can lead to malignant transformation
o Most common = columnar to squamous (ex. respiratory tract in response to chronic irritation)
o Usually comes with a trade-off of functional loss in order to gain resistance to irritants
o Mechanisms of Metaplasia
Results from reprogramming of stem cells or undifferentiated mesenchymal cells
Differentiation due to signals by cytokines, growth factors, and ECM components in cell's environment
Vitamin A (retinoic acid) deficiency commonly linked to inc. metaplasia

Overview of Cell Injury and Cell Death

Due to inability to adapt to severe stress, exposure to inherently damaging agents, or intrinsic abnormalities
Reversible cell injury
o Functional and morphologic changes are reversible if damaging stimulus is removed
o Reduced oxidative phosphorylation > depletion of ATP
o Changes in ion concentration and water flux > cellular swelling
o May also see changes in mitochondria and cytoskeleton
Cell death
o Necrosis = accidental and unregulated form of cell death due to damage of cell membranes
Cellular components leak unexpectedly > inflammation reaction
Always pathologic
o Apoptosis = cell kills itself in response to DNA or protein damage beyond repair
Cellular components cleaned up w/o unexpected leak > no inflammation
Not necessarily pathologic

Causes of Cell Injury

Oxygen deprivation (hypoxia) > reduce aerobic oxidative respiration
Physical agents (mechanical trauma, burns, deep cold, changes in atmospheric pressure, radiation, electric shock)
Chemical agents and drugs
Infectious agents
Immunologic reactions (autoimmune disease or reactions to external agents)
Genetic derangements
Nutritional imbalance (calorie/vitamin deficiencies or hypercholesterolemia)

Morphologic Alterations in Cell Injury

Reversible Injury
o Cellular swelling
Result of failure of energy-dependent ion pumps > incapable of maintaining ionic and fluid homeostasis
Plasma membrane alterations (blebbing, blunting, loss of microvillie)
Mitochondrial changes (swelling and appearance of small amorphous densities)
Dilation of ER w/ detachment of polysomes
Nuclear alterations w/ disaggregation of granular and fibrillar elements
o Fatty change
Occurs in hypoxic injury and other toxic or metabolic injury
Appearance of lipid vacuoles in the cytoplasm

Necrosis
o Denaturation of intracellular proteins and enzymatic digestion of lethally injured cell
o Cells unable to maintain membrane integrity > contents leak out > elicit inflammation
o Necrotic cells show increased eosinophilia and may be replaced by myelin figures (from damaged cell membranes)
o Nuclear changes
Karyolysis = loss of DNA due to enzymatic degradation by endonucleases
Pyknosis = nuclear shrinkage
Karyorrhexis = pyknotic nuclear undergoes fragmentation
o Patterns of Tissue Necrosis
Coagulative necrosis
Injury degrades enzymes > blocks proteolysis of dead cells > preserved architecture
 Localized area of coagulative necrosis = "infarct"
Liquefactive necrosis
Digestion of dead cells > tissue transformed into liquid viscous mass ("pus")
Often manifested in the CNS
Gangrenous necrosis
Not a specific pattern, but used clinically to describe a limb (generally lower leg)
Bacterial infection superimposed > additional degradative enzymes > "wet gangrene"
Caseous necrosis
White, cheese-like appearance, common in tuberculous infection.
Necrotic area of fragmented/lysed cells and granular debris enclosed by border = "granuloma"
Fat necrosis
Refers to focal areas of fat destruction, but not a specific pattern
Results from release of activated pancreatic lipases into pancreas and peritoneal cavity
Liberated fatty acids combine with calcium > chalky-white areas of "fat saponification"
Fibrinoid necrosis
Usually seen in immune reactions involving blood vessels
Occurs when complexes of antigens/antibodies are deposited in walls of arteries
Immune complexes + fibrin that leaks from vessels > bright pink "fibrinoid"
Necrotic cells and cellular debris not promptly cleaned up > calcified > "dystrophic calcification"

Mechanisms of Cell Injury

Depletion of ATP
o Fundamental cause of necrotic cell death
o Major causes of depletion = reduced supply of oxygen/nutrients, mitochondrial damage, and action of some toxins
o Reduction in ATP by 5-10% has widespread effects on cellular system:
Na/K ATPase activity reduced > Na+ inside > isosmotic gain of water > cell swelling/dilation of ER
Inc. anaerobic glycolysis > lactic acid produced > drop in intracellular pH > decreased activity of enzymes
Failure of Ca++ pump > influx of Ca++ > damaging effects on cellular components
Detachment of ribosomes from RER > reduction in protein synthesis
Accumulation of misfolded proteins in ER > unfolded protein response > cell injury/death
Irreversible damage to mitochondria and lysosomal membranes > necrosis

Mitochondrial Damage
o Can be damaged by inc. cytosolic Ca++, ROS, and oxygen deprivation
o Consequences of mitochondrial damage:
Formation of mitochondrial permeability transition pore > failure of ox phos > drop in ATP > necrosis
Abnormal oxidative phosphorylation > ROS
Release pro-apoptotic proteins (cytochrome c) from between membranes > activate caspases > apoptosis

Influx of Calcium and Loss of Calcium Homeostasis

o Accumulation in mitochondria > opening of mitochondrial permeability transition pore > failure of ATP generation
o Activates enzymes w/ potentially deleterious effects (ex. phospholipases, proteases, endonucleases, ATPases)
o Induction of apoptosis by direct activation of caspases and by inc. mitochondrial permeability

Accumulation of Oxygen-Derived Free Radicals (Oxidative Stress)

o Related to chemical and radiation injury, ischemia-reperfusion injury, cellular aging, and microbial killing
o Unpaired electrons attack and modify adjacent molecules
o Oxidative stress = excess ROS due to inc. production or decreased scavenging
o Generation of free radicals
Reduction-oxidation reaction that occurs during normal metabolic processes
Absorption of radiant energy
Produced in activated leukocytes during inflammation
Enzymatic metabolism of exogenous chemical or drugs
Transition metals donate or accept free electrons during intracellular reactions
Nitric oxide (NO) from endothelial cells/macrophages/neurons can act as a free radical
o Removal of free radicals
Antioxidants (vitamins E and A) block formation or inactivate free radicals
Minimize action of transition metals by binding to storage/transport proteins
 Free radical-scavenging system (catalase, superoxidase dismutase, glutathione peroxidase)
o Pathologic effects of free radicals
Lipid peroxidation in membranes > extensive membrane damage
Oxidative modification of proteins > damage active sites, disrupt structural proteins
Lesions in DNA > implicated in cell aging and malignant transformations

Defects in Membrane Permeability

o Consistent feature of most forms of cell injury, except apoptosis
o Mechanisms of membrane damage
Reactive oxygen species
Decreased phospholipid synthesis (due to defective mitochondrial fxn or hypoxia > dec. ATP)
Inc. phospholipid breakdown (due to activation of Ca ++-dependent phospholipase) > accumulation of lipid
breakdown products > detergent effect on membranes
Cytoskeletal abnormalities > detachment of cell membrane from cytoskeleton > susceptible to rupture
o Consequences of membrane damage
Mitochondrial membrane damage > decrease ATP generation and release or pro-apoptotic proteins
Plasma membrane damage > loss of osmotic balance
Lysosomal membrane damage > leak RNases, DNases, proteases, phosphatases, glucosidases

Damage to DNA and Proteins

o DNA damage too severe to be repaired or too many improperly folded proteins > apoptosis

Reversible vs. Irreversible Injury

o Point of no return:
Inability to reverse mitochondrial dysfunction
Profound disturbances in membrane function
o Leakage of intracellular proteins into circulation > detect tissue-specific cellular injury and necrosis
Cardiac muscle = creatine kinase + troponin
Liver = alkaline phosphatase + transaminases

Clinicopathologic Correlations: Selected Examples of Cell Injury and Necrosis

Ischemic and Hypoxic Injury
o Cell injury from reduced blood flow (commonly due to mechanical arterial obstruction)
Hypoxia > loss of oxidative phosphorylation > decreases ATP generation
Swelling of mitochondria, Ca++ influx
Reversible if oxygen is restored
Persistent ischemia > irreversible injury (mitochondria + membrane damage) > necrosis
o Defense mechanism = hypoxia-inducible factor-1 > promotes angiogenesis + cell survival + anaerobic glycolysis
o Treatment of ischemic brain/spinal cord injuries = transient induction of hypothermia > reduce metabolic demand,
decrease cell swelling, suppress formation of free radicals, inhibit inflammatory response

Ischemia-Reperfusion Injury
o Reperfusion can paradoxically exacerbate injury and cause cell death
o Oxidative stress due to incomplete reduction of O2 by damaged mitochondria or action of oxidases
o Intracellular calcium overload due to cell membrane damage and ROS injury to SR
o Inflammation due to cytokines, adhesion molecules, and danger signals from dead cells
o Activation of complement system due to IgM binding to ischemic tissues (for unknown reason)

Chemical (Toxic) Injury

o Major limitation to drug therapy, especially damage to liver
o Mainly affects organs involved in absorption, excretion, or metabolism of chemicals
Direct toxicity (ex. cyanide poisons mitochondrial cytochrome oxidase)
Conversion to toxic metabolites and free radicals (ex. acetaminophen)

Apoptosis
Programmed cell death > intrinsic enzymes degrade nuclear DNA and cellular proteins > break up into apoptotic bodies
Dead cells and fragments are rapidly phagocytized > doesn't elicit inflammatory reaction
Causes of Apoptosis
 o Apoptosis in Physiologic Situations
Destruction of cells during embryogenesis
Involution of hormone-dependent tissues upon hormone withdrawal
Cell loss in proliferating cell populations (ex. lymphocytes that don't express useful antigen receptors)
Elimination of potentially harmful self-reactive lymphocytes
Death of host cells that have served their useful purpose (ex. neutrophils and lymphocytes)
o Apoptosis in Pathologic Conditions
DNA damage (ex. radiation, cytotoxic anticancer drugs, hypoxia)
Accumulation of misfolded proteins > ER stress (due to mutations in genes or extrinsic factors)
Cell death in certain infections (ex. adenovirus, HIV, viral hepatitis)
Pathologic atrophy in parenchymal organs after duct obstruction (ex. pancreas, parotid gland, kidney)

Morphologic and Biochemical Changes in Apoptosis

o Morphology
Cell shrinkage
Chromatin condensation**
Formation of cytoplasmic blebs and apoptotic bodies
Phagocytosis of apoptotic cells or cell bodies, usually by macrophages
Lack of inflammation
o Mechanisms of Apoptosis
Results from activation of caspases (normally present as zymogens that must be cleaved)
Activation of caspases depends on balance between pro-apoptotic and anti-apoptotic proteins
o The Intrinsic (Mitochondrial) Pathway of Apoptosis
Major mechanism of apoptosis
Increased permeability of mitochondrial outer membrane > release of pro-apoptotic cytochrome c
Release of pro-apoptotic proteins controlled by BCL2 family of proteins
Anti-apoptotic = BCL2, BCL-XL, and MCL1 > prevent leakage of cytochrome c
Pro-apoptotic = BAX, BAK > promote mitochondrial outer membrane permeability
Sensors = BAD, BIM, BID, Puma, Noxa (BH3-only proteins) > sense cellular stress/damage >
regulate balance between anti-apoptotic and pro-apoptotic proteins
Cytochrome c binds APAF-1 (apoptosis-activating factor-1) in cytosol > forms apoptosome hexamer
Apoptosome binds caspase-9 (initiator caspase) > cleaves > activates caspase-9 > cascade
o The Extrinsic (Death Receptor-Initiated) Pathway of Apoptosis
Due to engagement of plasma membrane death receptors
Best known death receptors = type 1 TNF receptor (TNFR1) and Fas (CD95)
FasL (ligand) on T cells that recognize self antigens (kill themselves) and on CTLs (kill infected cells)
FasL binds Fas > 3 receptors aggregate > FADD (death domain) > bind and aggregate caspase-8 or 10
Caspase-8 or 10 cleave one another > active caspase-8 > activate multiple executioner caspases
o The Execution Phase of Apoptosis
Two initiating pathways converge on a cascade of caspase activation
Executioner caspases = caspase-3 or 6
Cleave inhibitor of cytoplasmic DNase > allows cleavage of DNA
Degrade structural components of nuclear matrix > fragmentation of nuclei
o Removal of Dead Cells
Apoptotic bodies small enough to be phagocytized
Apoptotic cells secrete soluble factors > recruit phagocytes
Phosphotidylserine on inner membrane flips to outer surface > target for macrophages
Coated by thrombospondin (adhesive glycoprotein) > recognized by phagocytes
Coated by complement system C1q > recognized by phagocytes

Clinocopathologic Correlations: Apoptosis in Health and Disease

o Examples of Apoptosis
Growth factor deprivation > trigger intrinsic (mitochondrial) pathway due to dec. BCL2/XL and inc. BIM
DNA damage > apoptotis due to p53 tumor suppressor activating BAX/BAK and some BH3-only proteins
Protein misfolding > ER stress activates caspases if unable to deal w/ misfolded protein accumulation
Cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor), Tay-Sachs disease
(hexosaminidase ), 1-antitrypsin deficiency (1-antitrypsin), Creutzfeldt-Jacob disease (prions),
Alzheimer disease (A peptide)
 Apoptosis induced by TNF receptor family > elimination of self lymphocytes and autoimmune diseases
Cytotoxic T lymphocyte-mediated apoptosis > CTL secrete granzymes that activate cellular caspases
o Disorders Associated w/ Dysregulated Apoptosis
Defective apoptosis and increased cell survival
Mutation in TP53 > defective DNA repair > accumulations of mutations > cancer
Failure to eliminate self reactive lymphocytes > autoimmune disease
Increased apoptosis and excessive cell death
Neurodegenerative disease > mutations and misfolded proteins > apoptosis
Ischemic injury (ex. MI)
Death of virus-infected cells in viral infections

Necroptosis
o Resembles necrosis = loss of ATP, cells/organelles swell, generate ROS, release lysosozymes, rupture membrane
o Resembles apoptosis = triggered by genetically programmed signal transduction (but caspase-independent)
o TNF > TNFR1 > recruit receptor associated kinase 1 and 3 (RIP1 and RIP3) into multimer > metabolic alterations
o Occurs during formation of bone growth plate, cell death in steatohepatitis, acute pancreatitis, reperfusion injury,
and neurodegenerative diseases (ex. Parkinsons)
Pyroptosis
o Another form of programmed cell death, accompanied by release of fever inducing IL-1
o Cytoplasmic immune receptors activate inflammasome > activate caspase-1 > cleaves IL-1 precursor
o Caspase-1 and 11 also induce death of the cells
o Unlike apoptosis, involves swelling, loss of plasma membrane integrity, and release of inflammatory mediators

Autophagy
Cell eats its own contents by delivering cytoplasmic material to lysosome for degradation
o Chaperone-mediated autophagy: direct translocation across lysosomal membrane by chaperone proteins
o Microautophagy: inward invagination of lysosomal membrane for delivery
o Macroautophagy (aka autophaghy): sequestration and transportation of portions of cytosol in double-membrane
bound autophagic vacuole (autophagosome)
States of nutrient deprivation > starved cell lives by cannibalizing itself and recycling the digested content
LC3 (light chain 3) protein targets protein aggregates and worn out organelles for loading into autophagosome
Autophagy in disease
o Cancer = autophaghy can both promote and defend against cancers
o Neurodegenerative disorders = dysregulation of autophagy (Alzheimer = accelerated, Huntington = impaired)
o Infectious diseases = pathogens degraded by autophaghy > delivered to APCs
o Inflammatory bowel disease = linked to Crohn diseasea and ulcerative colitis

Intracellular Accumulations
Lipids
o Steatosis (Fatty Change)
Abnormal accumulation of triglyceride within parenchymal cells
Often seen in liver, but also occurs in heart, muscle, and kidneys
Caused by toxins, protein malnutrition, diabetes mellitus, obesity, and anorexia
Most commonly due to alcohol abuse and nonalcoholic fatty liver disease (diabetes/obesity) in US
o Cholesterol and Cholesterol Esters
Atherosclerosis = macrophages in intimal layer of aorta and large aa. filled w/ lipid vacuoles (foam cells)
Xanthomas = foam cells in CT of skin and tendons
Cholesterolosis = cholesterol-laden macrophages in lamina propria of gallbladder
Niemann-Pick disease, type C = lysosomal storage disease > cholesterol accumulation in multiple organs
Proteins
o Appear as eosinophilic droplets, vacuoles, or aggregates in cytoplasm
o Reabsorption droplets in proximal renal tubules associated w/ proteinuria
o Overproduction of normal proteins > "Russell bodies" (hugely distended ER)
o Defective intracellular transport and secretion of critical proteins (misfolded proteins aren't excreted)
o Accumulation of cytoskeletal proteins (ex. neurofibrillary tangle in Alzheimer disease)
o Proteinopathies = aggregations of abnormal proteins deposited in tissues > interfere w/ fxn (ex. amyloidosis)
Hyaline Change
 o Homogenous, glassy, pink appearance on H&E stain
o Produced by a variety of alterations
o Intracellular accumulation of protein = reabsorption droplets, Russell bodies, alcoholic hyaline
o Extracellular = walls of arterioles in long-standing HTN and diabetes mellitus
Glycogen
o Excessive intracellular deposits seen in pts w/ abnormality in either glucose or glycogen metabolism
o Masses appear as clear vacuoles in the cytoplasm
o Diabetes mellitus = glycogen found in renal tubular epithelial cells, within liver cells, cells of islets of Langerhans,
and heart muscle cells
o Accumulates in cells w/ glycogen storage diseases (aka glycogenoses) > cell injury/death
Pigments
o Exogenous Pigments
Most common = carbon
Inhaled > picked up by alveolar macrophages > transported to regional lymph nodes
Results in blackening of lungs (anthracosis) and involved lymph nodes
o Endogenous Pigments
Lipofuscin (lipochrome aka wear-and-tear pigment) = indicative of free radical injury
Melanin = only endogenous brown-black pigment
Hemosiderin = hemoglobin-derived, golden yellow/brown, granular or crystalline, storage form of iron

Pathologic Calcification
Dystrophic Calcification
o Encountered in areas of necrosis
o Appear as fine, white granules or clumps, intracellularly and/or extracellularly
o Over time, may form "heterotropic bone" in the focus of calcification
o Seeded crystals are encrusted by mineral deposits > progressive accumulation of outer layers "psammoma bodies"
o Serum calcium is normal
Metastatic Calcification
o May occur in normal tissues whenever there is hypercalcemia
Increased secretion of parathyroid hormone (parathyroid tumors, ectopic PTH secretions due to
malignant tumros) > bone resorption
Resorption of bone tissue due to bone marrow tumors (multiple myeloma, leukemia), diffuse skeletal
metastasis (breast cancer), accelerated bone turnover (Paget disease), or immobilization
Vitamin D-related disorders (vitamin D intoxication, sarcoidosis, Williams syndrome)
Renal failure > retention of phosphate > hyperparathyroidism
Less common = aluminum intoxication, milk-alkali syndrome (excess ingestion of Ca++ and antacids)
o Principally affects interstitial tissues of gastric mucosa, kidneys, lungs, systemic arteries, and pulmonary vv.
o May occur as noncrystalline amorphous deposits or as hydroxyapatite crystals

Cellular Aging
Progressive decline in cellular function and viability caused by genetic abnormalities
Accumulation of cellular and molecular damage due to exposure to exogenous influences
DNA damage
o Due to carcinogen exposure, sporadic errors, ROS > defective DNA repair > DNA damage persists and accumulates
o Ex. Werner syndrome = defective DNA helicase (used in repair) > premature aging
o Ex. Bloom syndrome and ataxiatelangiectasia = defective repair proteins > premature aging
Cellular senescence
o Cells have a limited capacity for replication > arrested in nondividing state after a fixed number of divisions
o Telomere attrition (progressive shortening) > cell cycle arrest
Due to inactivation of telomerase (normally maintains telomere length)
Cancer cells show reactivation of telomerase > proliferate indefinitely
o Activation of tumor suppressor genes (CDKN2A locus)
p16 aka INK4a = controls G1 and S phage progression > protects cells from uncontrolled division signals
Defective protein homeostasis
o Maintain proteins in folded conformations (chaperones) + degrade misfolded proteins (autophagy or ubiquitin)
o Both folding and degradation are impaired w/ aging
Deregulated nutrient sensing
o Eating less increases longevity (caloric restriction increases life span in all species)
o Insulin and insulin-like growth factor 1 (IGF-1) signaling pathway = reduced by caloric restriction
IGF-1 produced in response to growth hormone secretion by the pituitary
IGF-1 informs cells of availability of glucose > promote anabolic state > cell growth and replication
Works via AKT and mTOR kinases (mTOR can also be inhibited by rapamycin)
o Sirtuins (NAD-dependent protein deacetylases) = increased by caloric restriction
Adapt bodily functions to environmental stresses (ex. food deprivation and DNA damage)
Promote expression of genes that increase longevity
Inhibit metabolic activity
Reduce apoptosis
Stimulate protien folding
Inhibit harmful effects of ROS
Also increase insulin sensitivity and glucose metabolism