Far Eastern University – Nicanor Reyes Medical Foundation CELLULAR RESPONSES TO STRESS AND NOXIOUS STIMULI

Pathology A – Cellular Responses to Stress and Toxic Insults:

Adaptation, Injury, and Death.
Pedrito Tagayuna M.D.

PATHOLOGY
 Study of structural, biochemical, and functional changes in
tissues, and organs that underlie disease.
 Pathos (Gk.) – Suffering.
 Divisions of Pathology:
 General Pathology
- Concerned with common reactions of cells and tissues to
injurious stimuli.
- Such reactions are not tissue specific.
 Systemic Pathology
- Examine alterations and underlying mechanisms in organ
specific diseases.

Four aspects of the disease process
ETIOLOGY PATHOGENESIS
MORPHOLOGIC CLINICAL
CHANGES MANIFESTATION
1.) ETIOLOGY
 The cause of the disease, which can be grouped into 2 classes:
 Genetic – inherited mutations and disease-associated gene
variants or polymorphisms.
 Acquired – Infectious, Nutritional, Chemical or Physical.
 Most of the common afflictions can be considered multifactorial
wherein various external triggers can affect genetically
susceptible individuals.
2.) PATHOGENESIS
 Sequence of cellular, biochemical, and molecular events that
follow the exposure of cells or tissues to an injurious agent.
 One of the main domains of pathology.
3.) MORPHOLOGIC CHANGES
 Structural alterations in cells or tissues that are either
characteristic of the disease itself or diagnostic of an etiologic
process.
 Morphologically identical lesions can be differentiated by
molecular analysis such as in cases of tumors.
4.) CLINICAL MANIFESTATIONS (Functional Derangements)
 End results of genetic, biochemical, and morphologic changes in
the tissues.
 Symptoms and signs of the disease.
 Clinical course and outcome.
 Rudolf Virchow: “Virtually all forms of disease start with
molecular or structural alterations in cells.”
 Cells able to handle physiologic demands and maintain
homeostasis.
 Adaptation is a reversible functional and structural response to
change in physiologic states (e.g pregnancy) or in some
pathologic stimuli, during which altered steady states are already
achieved.
 Adaptive changes can be in the form of:
 Hypertrophy
 Hyperplasia
 Atrophy
 Metaplasia
 If the injury is mild and the stimuli are removed, reversible cell
injury will happen and it can return to normal state so long that
the noxious stimuli are removed.
 If limits of adaptive responses are exceeded, irreversible cell
injury will happen and will progress to cell death.
 Cell death is the end result of progressive cell injury which may
be due to ischemia, infection, toxin, mechanical stressors or
even some physiologic needs (e.g. embryogenesis).
 There are two principal ways of cell death:
 Apoptosis
 Necrosis
ADAPTATIONS OF CELLULAR GROWTH AND DIFFERENTIATION
 Cellular adaptations are reversible changes in the cell.
1.) HYPERTROPHY
 Increase in the size of cells that result in an overall increase in
the size of the organ.
 There are no new cells produced, the cells just get larger.
 This is due to increased synthesis and assembly of intracellular
structural components.
 More prominent in non-dividing cells because they cannot
undergo hyperplasia.
 Hypertrophy can be physiologic or pathologic.

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 2.) HYPERPLASIA
 Increase in the number of cells in an organ or tissue.
 Frequently occurs together with hypertrophy in cells capable of
mitotic division.

Physiologic Hyperplasia
 Occurs due to actions of hormones or growth factors.
 Occurs when there is increased functional capacity or
compensatory increase after damage.
 Hormonal Hyperplasia: proliferation of glandular epithelium of
 Cells hypertrophy due to increased functional demands. the female breast during pregnancy.
 Stimulation of hormones and growth factors.  Compensatory Hyperplasia: Liver regeneration after
 Most common stimulus for muscle hypertrophy is increased hepatectomy.
workload (e.g. cardiac and skeletal muscles)
 Uterine Hypertrophy is a good example of hormone-induced Pathologic Hyperplasia
hypertrophy during pregnancy. (Physiologic Hypertrophy)  Occurs when there is an excessive or abnormal action of
 Faulty valves or increased hemodynamic load (e.g. Hypertension) hormones or growth factors.
is a stimulus for Cardiac hypertrophy. (Pathologic Hypertrophy)  Ex:
 Endometrial Hyperplasia – due to a stop in rising levels of
Mechanism of Hypertrophy progesterone.
 Increased production of cellular protein.  Benign Prostatic Hyperplasia – abnormal hormonal
 Myocardial Hypertrophy biochemical mechanisms: stimulation by androgens.

 Hyperplasia is different from cancer, but it constitutes a fertile

soil where cancer proliferation and malignancies may arise.
 Hyperplasia is also a characteristic response to papillomaviruses.
 Viral proteins can interfere with host proteins that regulate cell
proliferation.

Mechanism of Hyperplasia
 Growth factor driven cell proliferation.

3.) ATROPHY
 Reduction in the size of the organ.

Physiologic Atrophy:
 Integrated actions of mechanical sensors (which are  Such as during embryogenesis and developmental pathways.
triggered by increased workload), growth factors, and
vasoactive agents. Pathologic Atrophy:
 These three signals from the plasma membrane trigger two Decreased Workload
signal transduction pathways:  Initially the decrease in cell size is reversible as long as the disuse
a.) Phosphoinositide 3-kinase – important in physiologic. is not prolonged.
b.) G-Protein coupled messenger – important in pathologic.  Prolonged disuse can lead to permanent atrophy such as in
 The signaling pathways activate set of transcription factors: muscle atrophy.
a.) GATA4 Loss of Innervation (Denervation atrophy)
b.) NFAT (Nuclear Factor of Activated T-Lymphocyte)  Damage to nerve leads to disuse of muscles resulting to atrophy.
c.) MEF2 (Myocyte Enhancer Factor 2) Diminished Blood Supply
 Gradual decrease in blood supply could lead to atrophy.
 Hypertrophy is also associated with the shift of adult proteins to  Senile atrophy – due to atherosclerotic plaques.
neonatal proteins. Inadequate Nutrition
 Example in muscle hypertrophy, α-isoform of myosin heavy  Protein wasting (Marasmus)
chain is transformed to β-isoform in hypertrophic conditions.  Muscle Wasting (Cachexia)
 Genes expressed only during early development is reexpressed  TNF suppresses appetite and causes lipid depletion.
in hypertrophy.

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Loss of Endocrine Stimulation
 Prominent on hormone dependent organs such as the breast
and uterus.
Pressure
 Tissue compression for any length of time can cause atrophy.
 Benign tumors can compress vital surrounding tissues.
 Atrophic tissues have decrease cell size and organelles which
results to less metabolic requirements.
 In atrophic muscle, there may be fewer mitochondria and less
ribosome in the ER.
 Early in the process, atrophic tissues have diminished function
but cell death is not prominent.
Mechanism of Atrophy
 Decreased Protein Synthesis due to decreased metabolic activity.
 Increased Protein Degradation.
 Occurs mainly by Ubiquitin Proteosome Pathway.
 Ubiquitin (76-AA chain) tag cellular proteins which then become
target for proteasomes for degradation.
 Accelerated proteolysis.
 Autophagy is also prominent in atrophy, leaving some cell debris
and increase in autophagic vacuoles (e.g. Lipofuscin granules).
4.) METAPLASIA
 Reversible change one differentiated cell type is replaced by
another cell type.
 Adaptive response wherein one cell type is replaced by another
cell type because the latte can handle the stress compared to
the first one.
Columnar to Squamous (Squamous Metaplasia)
 Columnar ciliated epithelium of the respiratory tract is replaced
with stratified squamous epithelium in response to chronic
irritation.
 Prominent in smokers.
 It is also present in secretory epithelium of glandular organs
undergoing squamous metaplasia in response to calculi
formations.
 Vitamin A deficiency also causes squamous metaplasia in the
respiratory epithelium.
 The stratified squamous is able to survive circumstances that the
columnar epithelium may have succumbed to.
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 However, the squamous epithelium removes the muco-ciliary
clearance system of the respiratory epithelium, rendering it
available to infectious agents.
 Persistent squamous metaplasia can lead to squamous cell
carcinoma of the respiratory tract.
Squamous to Columnar
 Present in Barrett Esophagus.
 The squamous lining of the esophageal epithelium is replaced by
columnar in response to gastric acid reflux.
 Cancer cells can also arise in these areas (adenocarcinomas)
Connective Tissue Metaplasia
 Formation of cartilage, bone, or adipose tissue in areas where
they are normally not present.
 Myositis ossificans – formation of bone in muscle fibers which
normally occur after intramuscular hemorrhage.
Mechanism of Metaplasia
 It is the result of reprogramming of stem cells.
 The precursor cells differentiate along new pathways driven by
cytokines, growth factors.
 Retinoic Acid deficiency can lead to transcription dysfunction
since it is a lipophilic substance and a direct regulator of gene
transcription.
OVERVIEW OF CELL INJURY AND DEATH
Reversible Cell Injury
 Injury is reversible if the damaging stimuli are removed.
 Reduced oxidative phosphorylation results to reduced ATP.
 Reduced ATP makes the Na-K pump to stop working.
 There will be Na influx and K efflux, together with Na, water will
go inside the cell and cause cellular swelling.
Cell Death (Irreversible Cell Injury)
 Continuous damaging stimuli will result in irreversible cell death.
 Cell Death can happen via two ways:
 Apoptosis
 Necrosis
 The two processes vary in their morphology, mechanism, and
roles in physiology and diseases.
Necrosis Nutritional Imbalances
 Accidental and unregulated form of cell death.  Protein-Calorie Deficiencies.
 Results from damage to cell membrane and loss of ion  Vitamin Deficiencies.
homeostasis.  Nutritional Excess.
 Lysozymes digest the cytoplasm and cytoplasmic content leak
which often elicit inflammatory reactions. MORPHOLOGIC CHANGES IN CELL INJURY
 Necrosis is commonly encountered in injuries.

Apoptosis
 Occurs in cells when DNA damage is beyond repair.
 Programmed cell death.
 Nuclear dissolution and cell fragmentation without leaking of the
contents.
 No inflammatory reaction.

 Necrosis is always a pathologic process.

 Apoptosis can be physiologic or pathologic.
 In some cases, a different set of genes can trigger a necrosis-like
mechanism of cell death, it is called NECROPTOSIS.
CAUSES OF CELL INJURY
Oxygen Deprivation
 Hypoxia is a deficiency of oxygen.
 It causes cell injury by reducing aerobic oxidative mechanisms.
 Causes of Hypoxia:
 Ischemia – reduced blood flow (Atherosclerotic plaques)
 Inadequate oxygenation of blood (Cardiorespiratory failure)
 Decreased oxygen carrying capacity (Carbon monoxide
Poisoning – formation of irreversible monoxyhemoglobin.)
Physical Injuries
 Mechanical Trauma.
 Extreme Temperatures.
 Radiation, Pressure, Electric Shock.
 All stresses and noxious influences exert their effects first at the
molecular or biochemical level.
 There is a time lag between the stress and the morphologic
changes.
 Ultrastructural and Biochemical changes can be seen in minutes
or hours.
 However, it takes days to weeks to see microscopic and gross
morphological changes.
Morphologic features of Reversible Injury
1.) Cellular Swelling – because of reduced oxidative
phosphorylation, there is no ATP to be used in the Na-K pump,
allowing sodium influx with water causing swelling.
2.) Fatty Change – Occurs in hypoxic injuries presents as lipid
vacuoles in the cytoplasm, prominent in cells which are dependent
on lipid metabolism (e.g. hepatocytes and cardiac myocytes)

Chemical Agents and Drugs

 Can cause cell injury by deranging electrolyte balance.
MORPHOLOGY OF CELLULAR SWELLING
 Poisons, Air Pollutants, High conc. of oxygen.
 Causes some pallor (paleness)
 Increased Turgor
Infectious Agents
 Increase in the weight of the organ
 Virus, Bacteria, Fungi, Tapeworms.
 Microscopically, small clear vacuoles and eosinophilia.

Immunologic Reactions
1.) Plasma Membrane Alteration
 Endogenous self-antigens can be responsible for autoimmune
 Blebbing, Blunting, Loss of Microvilli.
diseases.
2.) Mitochondrial Changes
 Immune reaction to external stimuli can also cause cell injury.
 Swelling and Small Amorphous Densities.
3.) Dilation of the ER
Genetic Derangements
 Detachment of polysomes, presence of intracytoplasmic myelin
 Extra chromosomes (Down Syndrome)
figures.
 Singe Base Pair Substitution (Sickle Cell Anemia)
4.) Nuclear Alterations
 Enzyme defects in inborn errors of metabolisms (PKU)
 Diaggregation of granular and fibrillary elements.
 Accumulation of damaged DNA and misfolded proteins
 All of them can trigger cell death.

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A Normal kidney tubules with viable epithelial cells.
B Early reversible ischemic injury, with surface blebs and
increased eosinophilia.
C Necrotic tubule. (Irreversible)
Loss of nuclei, fragmentation, and cytoplasmic leakage
NECROSIS VS APOPTOSIS
Morphologic Changes in Necrosis
 Result of denaturation of intracellular proteins and enzymatic
digestion of the lethally injured cell.
 Unable to maintain membrane integrity and contents leak out.
 Lysosomes of the cell itself leak their own enzymes combined
with lysozymes from the leukocytes causes degradation of the
cell.
 Increased Eosinophilia: since the proteins are denatured and
RNA in the cytoplasm is lost, which are basophilic, there is
nothing to absorb the blue dye.
 What remains is the leaked cytoplasm which is eosinophilic and
absorbs all the red dye.
 Myelin Figures: Whorled Phospholipid masses derived from
damaged cell membranes.
 Cytoplasm becomes vacuolated having a moth-eaten
appearance.
 Cells have more glassy homogenous appearance due to lost
glycogen particles.
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 Fatty acids from the cell can be degraded by lipases and combine
with calcium (saponification) to form calcium soaps and
ultimately become calcified.
 Electron Microscopy:
 Discontinuation of the plasma and organelle membranes.
 Dilation of the mitochondria with large amorphouse
densities.
 Amorphous debris, myelin figures, aggregates.
 Nuclear Change:
 Karyolysis – Basophilia of the chromatin and enzymatic
degradation of the DNA.
 Pyknosis – Nuclear Shrinkage and Increased Basophilia.
 Karyorrhexis – Pyknotic nucleus undergoes fragmentation.
PATTERNS OF NECROSIS
Coagulative Necrosis
 Preserved architecture of the dead tissue.
 Denatures all proteins including enzymes.
 Since even the cellular enzymes are denatured, the cells are
removed by phagocytosis of leukocytes.
 Ischemia secondary to blood vessel injury is the most common
cause of coagulative in all organs EXCEPT the brain.
 Localized coagulative necrotic area is called an Infarct.
Liquefactive Necrosis
 Digestion of the dead cells.
 Transformation of the solid tissue to liquid viscous mass.
 Bacterial or Fungal infections is the most common cause of
liquefactive necrosis.
 There is accumulation of dead leukocytes or pus.
 Hypoxic death of cells in the CNS is considered liquefactive.
Gangrenous Necrosis
 Not a specific pattern of necrosis.
 Commonly used on the limbs, especially the lower limbs.
 The area usually loses blood supply and undergoes necrosis
(coagulative).
 When bacterial infection is superimpoased, there is more
liquefactive then coagulative necrosis (wet gangrene).
Caseous Necrosis
 Tuberculous Infections is the most common cause.
 “Cheese-like” appearance.
 Necrotic area appears as structure less collection of lysed cells
and amorphous granular debris.
 It is usually enclosed within a distinctive inflammatory border,
common appearance of granulomas.
Fat Necrosis
 Not a specific pattern of necrosis.
 Refers to focal areas of fat destruction, primarily due to release
of pancreatic lipases (Acute Pancreatitis).
 The lipase splits TAG and Free FA’s combine with Calcium
(Saponification).
 In histologic preparations, it appears as shadowy outlines of
necrotic fat cells, with basophilic calcium deposits, surrounded
by inflammatory reaction.
Fibrinoid Necrosis
 Immune Reactions involving Blood vessels.
 Antigen-antibody complexes that get deposited in the arterial
wall. The immune complex leak out with fibrin.
 Bright pink amorphous appearance: Fibrinoid.
 Vasculitis Syndrome.
MECHANISM OF CELL INJURY
1.) Cellular response to injurious stimuli depends on the nature,
duration, and severity of the injury.
2.) The consequences of cell injury depend on the type, state, and
adaptability of the injured cell.
3.) Cell injuries results from different biochemical mechanisms
acting on several essential cellular components.
BIOCHEMICAL MECHANISMS IN RESPONSE TO INJURY:
A.) DEPLETION OF ATP
 Associated with both hypoxic and chemical injury.
 Major pathway is oxidative phosphorylation of ADP.
 Resulting to reduction of oxygen by the ETC.
 Second pathway is glycolytic pathway, ATP generation in the
absence of oxygen via hydrolysis of glycogen stores.
 Depletion of ATP to 10-15% causes the following:
 Energy-dependent pumps are reduced, specifically the Na-
K pump. Without this transport, Na will go in, and K will go
out of the cell. Together with Na, water will follow and
cause cell swelling.
 Cellular Energy Metabolism is altered, cell shifts first to
glycolytic pathway, after the depletion of glycogen stores, it
shifts to anaerobic glycolysis but results to accumulation of
lactic acid.
 Failure of Ca++ pump, causes calcium influx.
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 Disruption of protein synthesis apparatus.
 Accumulation of misfolded proteins in the ER resulting to
Unfolded Protein Response that can culminate cell death.
 Damage to lysosomal and mitochondrial membrane
resulting to cell death.
B.) MITOCHONDRIAL DAMAGE
 Mitochondria can be damaged by:
 Increased Cytosolic Ca++
 ROS (Reactive Oxygen Species)
 Oxygen Deprivation
 There are 3 major consequences of mitochondrial damage:
1. Mitochondrial Permeability Transition Pore
- Formation of this high conductance channel leads to the
loss of the mitochondrial potential.
- Failure of oxidative phosphorylation.
- Progressive depletion of ATP.
- Cyclophilin D – a protein component of the mitochondrial
transition pore, targeted by immunosuppressive drugs (e.g.
Cyclosporin) which prevent opening of the transition pores.
2. Abnormal Oxidative Phosphorylation
- Formation of Reactive Oxygen Species (ROS)
3. Activation of Apoptotic Pathways
- Apoptotic proteins are present in the intermembrane
space that can trigger cell death when leaked in the cytosol
(e.g. Cytochrome C and Caspases).
C.) INFLUX OF CALCIUM AND LOSS OF CALCIUM HOMEOSTASIS
 Cytosolic free Ca++ is normally maintained at very low
concentrations ~0.1µmol compared with extracellular levels of
1.3mmol.
 Increased intracellular Ca++ causes damage by:
 Accumulation of Ca++ in the mitochondria increases its
permeability, resulting to failed ATP generation.
 Activates intracellular enzymes (nucleases, proteases, etc.)
 Activates the caspase cascade system results to apoptosis.
D.) ACCUMULATION OF OXYGEN-DERIVED FREE RADICALS
(OXIDATIVE STRESS)
 Free Radicals are chemical species that have a single unpaired
electron in an outer orbit.
 These unpaired electrons are highly reactive and can modify
adjacent molecules.
 Reactions most of the time are autocatalytic.
 Reactive Oxygen Species (ROS) are produced normally during
respiration but degraded and removed by the cellular defense
system.
 Increased Production and Decreased scavenging of ROS can lead
to Oxidative Stress.
 Oxidative stress has been implicated in variety of pathologic
processes (e.g. Aging, Cancer, Cell Injury, and neurodegenerative
diseases).
 ROS is produced in large amount by neutrophils and
macrophages to kill bacteria.
Generation of Free Radicals
 Redox Reactions during normal metabolic processes, O2 is
reduced by the transfer of four electrons to H2 to generate 2
water molecules. In this process small amounts of partially
reduced intermediates are produced in the forms of:
 Hydrogen Peroxide
 Superoxide Anion
 Hydroxyl Ions
 Absorption of radiant energy: Ionizing radiation can convert
water to hydroxyl ions and H free radicals.
 Inflammation: Leukocytes produce large amounts of ROS,
carried out by the NADPH oxidase in the plasma membrane.
Xanthine Oxidase and other intracellular enzymes can also
generate ROS.
 Enzymatic Metabolism of Drugs: Can generate free radicals but
not ROS (e.g CCl4 can generate CCl3).
 Transition Metals: Iron and Copper can donate free electrons,
since they need to be reduced to be absorbed (Ferric to Ferrous).
It is explained by Fenton’s Reaction:
 Nitric Oxide: Produced by endothelial cells and macrophages can
be converted to highly reactive Peroxynitrite Anion.
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Removal of Free Radicals
 Free radicals are generally unstable and decay spontaneously.
 Antioxidants, such as Vitamin A and E, ascorbic acid, and
glutathione block free radical formation.
 Binding of metals to their transport protein, such as Ferritin and
Ceruloplasmin, can decrease the reaction of Iron and Copper
with Free radicals.
 Enzyme located near the generation sites of ROS can help
degrade them:
 Catalase – degrades H2O2 to H2O and O2.
 Superoxide Desmutase – degrades Superoxide to
Hydrogen Peroxide (H2O2). Has 2 forms:
- Manganese SOD: located in the mitochondria.
- Copper-Zinc SOD: located in the cytosol.
 Glutathione Peroxidase – catalyzes free radical
breakdown by recycling glutathione. Glutathione serves as
a donor of electron.
Pathologic Effects of Free Radicals
1.) Lipid Peroxidation of the Membrane
 Double bonds in unsaturated FAs of the membrane are attacked
by O2 derived free radicals (autocatalytic).
2.) Oxidative Modification of Proteins
 Formation of disulfide linkages.
 Oxidation of protein backbones.
 Disrupt conformational structure of proteins resulting to
proteosomal degradation.
3.) Lesions in the DNA
 Single and Double Strand breaks.
 Formation of adducts
 Cross-linking
 Malignant transformation.
E.) DEFECTS IN MEMBRANE PERMEABILITY
 Loss of membrane’s selective permeability.
Mechanism of Membrane Damage:
 Result in ATP depletion and Ca-mediated activation of
phospholipases.
 Can also be a direct damage from bacterial toxins, viral proteins,
lytic complement, physical, and chemical agents.
 ROS can cause lipid peroxidation.
 Decreased Phospholipid Synthesis due to defective
mitochondrial function and decreased ATP affecting energy-
dependent biosynthetic pathways.
 Increased Phospholipid Breakdown due to activation of Ca-
dependent phospholipases. Unesterified free fatty acids in the
membrane have a detergent effect.
 Cytoskeletal Abnormalities: An increase in cytosolic calcium can
activate proteases that damage the cytoskeleton and detach
them from the membrane making it susceptible to stretch and
rupture.
Consequences of Membrane Damage:
 Mitochondiral Membrane Damage: Opening of the permeability
pores.
 Plasma Membrane Damage: Loss of osmotic balance, influx of
fluids, and leak of metabolites.
 Injury to Lysosomal Membranes: Since the lysosomes are also
membrane bound, phospholipases can degrade their membrane
and leak their enzymes in the cytoplasm.
F.) DNA AND PROTEIN DAMAGE
 DNA Damage beyond repair results to cell death.
 Same with proteins, too much misfolded proteins can trigger cell
death.
 Irreversible due to:
 Mitochondrial dysfunction
 Membrane Disturbances (leakages)
CLINICOPATHOLOGIC CORRELATIONS: Selected examples of Cell
Injury and Necrosis
1.) ISCHEMIA AND HYPOXIC INJURY
 Ischemia is the most common type of cell injury.
 Results from hypoxia induced by reduced blood flow (most
commonly by mechanical obstruction in the arteries)
 Oxidative phosphorylation and glycogen stores are
compromised.
 Ischemia tends to cause more rapid and severe cell and tissue
injury than hypoxia without ischemia.
Mechanism of Ischemia and Hypoxia
 Aside from the previous mentioned mechanism of hypoxia,
cytoskeletons disperse and result in loss of ultrastructural
features such as microvilli.
 Formation of membrane blebs.
 Myelin figures also form from derived degenerating plasma
membrane.
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 Mitochondria are usually swollen.
 Endoplasmic Reticulum remains dilated.
 However, if oxygen is restored cell injury is reversible. But if
ischemia persists, it can lead to irreversible cell injury:
 Severe mitochondrial swelling.
 Extensive damage to the plasma membrane (myelin figures).
 Swelling of the lysosomes.
 Death is mainly by necrosis but apoptosis can partially contribute
due to activation of pro-apoptotic molecules from the leaky
mitochondria.
 Calcification via calcium soaps may occur.
 Hypoxia Inducibe Factor 1: a protective mechanism in response
to hypoxic stress that:
 Promote the new formation of blood vessels.
 Stimulate cell survival pathways.
 Enhance anaerobic glycolysis.
2.) ISCHEMIA REPERFUSION INJURY
 Restoration of blood flow to ischemic tissues can promote
recovery of cells but paradoxically can exacerbate the injury and
cause cell death.
 Clinically important in myocardial and cerebral infarctions.
 Mechanisms of new damaging process:
 Oxidative Stress – increased generation of ROS, it can also
be due to incomplete reduction of oxygen.
 Intracellular Calcium Overload – Ca influx can happen
during reperfusion and mediate mitochondrial damage.
 Inflammation:
- Danger signals from dead cells. (“Eat me” signals)
- Cytokines secreted by resident immune cells.
- Increased expression of adhesion molecules by hypoxic
parenchymal and endothelial cells can recruit circulating
neutrophils to reperfused tissue.
 Activation of the Complement System, IgM have a
propensity to deposit in ischemic tissues for an unknown
reason, upon reperfusion, complement system can bind to
this antibodies.
3.) CHEMICAL (TOXIC) INJURIES
 A frequent problem in clinical medicine.
 A major limitation to drug therapy.
 Toxic liver injury is the most frequent reason for stopping
therapeutic use or development of drugs.
Direct Toxicity
 Injures the cell directly.
 Mercury Chloride Poisoning, Hg binds to sulfhydryl groups of cell
membrane proteins that can inhibit ion transport. Greatest
damage on cells involved in absorption, excretion, and
concentration of chemicals. In HgCl2 poisoning, it affects GIT and
Kidney tissues.
 Cyanide Poisoning, CN binds to Complex 4 of the ETC inhibiting
oxidative phosphorylation inducing direct cell damage.
Indirect Toxicity
 Usually accomplished by the Cytochrome P450 in the smooth ER
of the liver.
 Formation of free radicals.
 CCl4 used in cleaning industry is converted by CytP450 to a free
radical CCl3.
 Acetaminophen, an analgesic drug, is also converted into a toxic
product during liver detoxification.
APOPTOSIS
 Tightly regulated suicide program.
 Apoptotic cells break up into fragments and form apoptotic
bodies.
 Plasma membrane of apoptotic cells remains intact but it is
altered to attract macrophages and phagocytize them before
their contents leak out.
 Genetically regulated process.
Causes of Apoptosis
Physiologic Apoptosis
 Destruction of cells during embryogenesis (implantation,
organogenesis, developmental pathways, and metamorphosis).
 Involution of hormone dependent tissue upon hormone
withdrawal:
 Endometrial cell breakdown during menstruation
 Ovarian Follicular Atresia during menopause.
 Prostatic Atrophy after castration.
 Cell loss in proliferating cell population
 Immature lymphocytes in bone and thymus
 B Lymphocytes in germinal centers
 Epithelial cells in intestinal crypts.
 Elimination of potentially harmful self-reactive T-lymphocytes
 Death of host cells after it has served its function.
 Neutrophil dies after acute inflammation.
 Lymphocytes at the end of an immune response.
Pathologic Apoptosis
 Apoptosis eliminates cells that are injured beyond repair,
without eliciting a host reaction.
 DNA damage from radiation or cytotoxic cancer drugs, the cell
triggers intrinsic mechanisms that induce apoptosis.
 Accumulation of misfolded proteins that may arise from
mutations in genes from free radical damage. This can cause
accumulation of misfolded proteins in the ER causing ER Stress.
 Infections induced by viruses can trigger apoptosis. It can also be
triggered by the host immune response. Cytotoxic T lymphocytes
can induce apoptosis.
 Atrophy after duct obstruction such as calculi or stone
formation in the ducts of the pancreas, parotid, and kidney.
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Morphologic Changes in Apoptosis
 Cell shrinkage, cell is smaller and cytoplasm becomes dense,
organelles become tightly packed.
 Chromatin Condensation most characteristic feature of
apoptosis wherein the chromatin aggregates peripherally.
 Formation of cytoplasmic blebs and apoptotic body’s extensive
surface blebbing results to fragmentation of the cytoplasm.
 Phagocytosis of the apoptotic fragments, rapidly ingested by
macrophages.
 Apoptotic cells appear as round or oval masses of intensely
eosinophilic cytoplasm with fragments of dense nuclear
chromatin.
Mechanism of Apoptosis
 Apoptosis results from the activation of enzymes called caspases
 They are cysteine proteases that cleave proteins after aspartic
residues.
 The process of apoptosis can be divided into two:
 Initiation Phase – some caspases become catalytically
active.
 Execution Phase – other caspases trigger degradation of
critical cellular components.
 Two distinct pathways of caspase activation:
 Mitochondrial (Intrinsic) Pathway
 Death Receptor (Extrinsic) Pathway
INTRINSIC PATHWAY OF APOPTOSIS
 Major pathway.
 Results from the increased permeability of the outer membrane.
 Cytochrome C, when released into the cytoplasm from the
mitochondria initiates apoptosis.
Anti-Apoptotic Proteins
 BCL2, BCL-XL, MCL1
 These reside in the outer membrane of the mitochondrial
membranes, as well as the cytosol and ER Membranes.
 These proteins keep the mitochondrial outer membrane
impermeable, preventing the leakage of cytochrome c.
Pro-Apoptotic Proteins
 BAX and BAK
 These proteins oligomerize within the outer membrane of the
mitochondria to promote permeability.
Sensors
 BAD, BIM, BID, Puma, Noxa (BH3-only proteins)
 Sensors of cellular stress and damage.
 Growth factors stimulate the production of anti-apoptotic
proteins preventing leakage of cytochrome c.
 However, when the DNA is damaged, BH3 proteins can detect it
and activate the pro-apoptotic proteins.
 Anti-apoptotic proteins’ synthesis may decline because of the
relative deficiency of survival signals.
 Once in the cytosol, cytochrome c, binds to a protein called
Apoptosis-Activating Factor 1 (APAF1) and form a wheel-like
hexamer called apoptosome.
 The complex binds to CASPASE-9 and initiates the cascade via
autoamplification, activating the other pro-caspases.
 Proteins such as Smac and Diablo bind and neutralize proteins
that function as physiologic inhibitors of apoptosis.
EXTRINSIC PATHWAY OF APOPTOSIS
 Initiated by engagement of plasma membrane death receptors
on a variety of cells.
 TNF-receptor family, contain a cytoplasmic domain involved in
protein-protein interaction called death domain.
 TNFR1 and Fas (CD95) are best known death receptors.
 Fas Ligand (FasL) for Fas receptor is expressed on T cells that
recognize self-antigens. Upon binding of 3 or more FasL to Fas
receptors, they are brought together and their death domain
form an adaptor protein that contains another death domain
called Fas-associated Death Domain (FADD).
 FADD binds inactive caspase 8 (caspase 10 in humans), when
multiple inactive caspase 8 molecules are brought to proximity,
they tend to cleave one another and activate the cascade.
 FLIP protein inhibits this pathway of apoptosis by binding to pro-
caspase 8 rendering it inactive. FLIP is produced by some viruses
to protect them from cell death.
 Combined activation of both pathways can be fatal to the cell.
EXECUTION PHASE OF APOPTOSIS
 Two initiating pathways converge to a cascade of caspase
activation (intrinsic – caspase 9; extrinsic – caspase 8 & 10)
 Executioner caspases such as caspase 3 and 6 act on many
cellular components (e.g. it can cleave an inhibitor of a
cytoplasmic DNase making it active).
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REMOVAL OF THE DEAD CELL
 Phosphatidylserine, a part of the plasma membrane, flips out
and expressed outside the plasma membrane, recognized by
many macrophages as an “eat me” signal.
 Thrombospondin coats most of the apoptotic bodies which is an
adhesion glycoprotein recognized by phagocytes.
 Cq1, a part of the complement system, can naturally coat the
apoptotic bodies for macrophages to recognize them.
CLINICOPATHOLOGIC CORRELATIONS: Apoptosis in Health and Disease.
Examples of Apoptosis
 Growth Factor Deprivation
- Triggered by intrinsic pathway of apoptosis.
- Hormone-sensitive cells deprived from relevant hormone.
- Lymphocyte not stimulated by antigens.
- Neurons deprived of nerve growth factor.
 DNA Damage
- Tumor suppressor gene (TP53) triggers apoptosis.
- TP53 stimulates the production of pro-apoptotic proteins and
BH3 proteins.
 Protein Misfolding
- Ubiquinated proteins are target for proteosomal degradation.
- Accumulation of misfolded proteins can trigger unfolded
protein response which activates protein signaling pathway
increasing the production of chaperones that enhance
proteosomal degradation.
- Unable to cope pu with misfolded protein activates the
caspases and induce apoptosis, this is called ER Stress.
 TNF-induced Apoptosis
- FasL on T cells eliminates lymphocytes that recognize self-
antigens.
- Mutations on Fas gene result in autoimmune diseases.
 Cytotoxic T-Lymphocyte Mediated Apoptosis
- CTLs recognize foreign antigens in infected host cells.
- CTLs release perforin molecule that forms pores allowing the
entry of Granzymes (Granule Serine Protease) in the cytosol ti
activate caspases,
Dysregulated Apoptosis
 Defective apoptosis, increased survival.
- TP53 mutations
- Autoimmune disorders
 Increased apoptosis, excessive cell death.
- Neurodegenerative diseases
- Ischemia
- Death of Virus-infected cell
NECROPTOSIS Steps in Autophagy
 Morphologically and biochemically, resembles necrosis.
 Triggered by genetic programmed signal transduction, like
apoptosis.
 Can be called as programmed necrosis.
 Driven passively by toxic or anoxic injuries.
 It does not result to caspase activation; hence it is sometimes
called caspase-independent programmed cell death.

Mechanism of Necroptosis
 Starts in a manner similar to extrinsic apoptosis via ligand-
receptor. 1. Formation of an isolation membrane called phagophore,
 Necroptosis involves 2 major unique kinases: derived from the ER.
 Repector associated kinase 1 and 3 (RIP1 & RIP3) 2. Elongation of the vesicle.
 When TNF binds to TNFR1, it recruits RIP1 and RIP3 molecules 3. Maturation of the autophagosome.
into a multiprotein complex that also contains caspase 8. 4. Fusion with the lysosomes.
5. Degradation of the content.
 However, the caspase is not activated, but the progress goes like
that of necrosis (ROS, lysosomal membrane destruction)
Roles of Autophagy
 Pathologic Necroptosis: Steatohepatitis, acute pancreatitis,
 Cancer: Both promote and inhibit cancer growth.
reperfusion injury, and neurodegenerative diseases.
 Neurodegenerative disorders:
 Physiologic Necroptosis: Formation of the mammalian bone
growth plate.  Alzheimer’s – formation of autophagosomes.
 Necroptosis also acts as a backup mechanism in host defense  Huntington’s D – Mutant ‘huntingtin’ (impaired
against viral infections that bypass the caspase system such as autophagy)
the cytomegalovirus.  Infection
 Shigella sp. And HSV-1
PYROPTOSIS  Macrophage deletion of ATG5, increases susceptibility to
 Accompanied by release of fever inducing cytokine IL-1 forming tuberculosis.
a multiprotein complex called inflammasome.  Inflammatory Bowel Diseases
 Inflammasomes activate caspase 1 (also known as Interleukin-1β  Crohn’s Disease
converting enzyme) which cleaves IL-β1 making it biologically  Ulcerative Colitis
active.
 Caspase 1 and caspase 11 induce cell death. INTRACELLULAR ACCUMULATION
 Unlike apoptosis, this present with cell swelling, loss of plasma  Intracellular accumulation of abnormal amounts of various
membrane integrity, and release of inflammatory mediators. substances may be harmless or associated with varying degrees
of injury.
AUTOPHAGY  Four main pathways of abnormal intracellular accumulation:
 A process where in a cell eats its own components. 1.) Inadequate Removal of a substance
 Involves the delivery of cytoplasmic materials to the lysosome  Defects in mechanisms of packaging and transport
for degradation  Ex: Hepatic Steatosis
 Three types:
 Chaperone-mediated 2.) Accumulation of an abnormal endogenous substance
- Direct translocation across the lysosomal membrane.  A result of genetic or acquired defects in its folding, packaging,
 Microautophagy and transport.
- Inward invagination of lysosomal membrane.  Ex: α1-antitrypsin
 Macroautophagy
- Major form of autophagy. 3.) Failure to degrade a metabolite
- Transport of portions of cytosol in a double membrane  Due to enzyme deficiencies
bound autophagic vaguole (autophagosome).  Ex: Storage diseases
 Autophagy is an evolutionarily conserved survival mechanisms:
 Used in states of nutrient deprivation 4.) Deposition and accumulation of an abnormal exogenous subs.
 Cells live by cannibalizing or recycling themselves  Cell has neither the enzymatic machinery to degrade the
 Controlled by the AGTS gene. substance, nor the ability to transport it to the other site
 Ex: Accumulation of Carbon or Silica particles

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LIPIDS
Steatosis (Fatty Changes)
 Describe abnormal accumulations of triglycerides within
parenchymal cells.
 Seen mainly in liver due to its site of fat metabolism.
 Caused by toxins, protein malnutrition, diabetes, obesity, and
anorexia.
Cholesterol and Cholesterol Esters
 Cholesterol is used in constructing the cell membrane but it is
sometimes seen in pathologic conditions:
 Atherosclerosis – plaque formation in the intimal layer and
smooth muscle layer of arteries filled with lipid vacuoles
producing yellow cholesterol-laden arethoma.
 Xanthoma – intracellular accumulation of cholesterol within
macrophages, seen in hyperlipidemic states.
 Cholesterolosis – accumulation of cholesterol-laden
macrophages in the gall bladder.
 Niemann-Pick disease type C – lysosomal storage disorder,
resulting to cholesterol trafficking and accumulation in different
organs.
PROTEINS
 Usually appear as rounded, eosinophilic droplets, vacuoles, or
aggregates in the cytoplasm.
 In electron microscopy: Amorphous, fibrillary, or crystalline.
 Caused by:
 Reabsorption droplets in the PCT
- Seen in renal disease associated with proteinuria.
- During heavy protein leakage, protein appears as pink,
hyaline substance seen in tubular cells.
 Russell Bodies
- When ER becomes hugely distended, producing large,
homogenous, eosinophilic inclusions.
 Defective intracellular transport
- In α anti trypsin deficiency, there is slow protein folding
leading to partially folded ones.
 Accumulation of cytoskeletal proteins
- Neurofibillary tangles
- Alcoholic hyaline is eosinophilic on cirrhotic patients.
 Aggregation of abnormal proteins.
- Amyloidosis.
- Proteinopathies or Protain-aggregation disorders.
HYALINE CHANGE
 Alterations within the cells or in the extracellular space.
 Gives homogenous, glassy, pink appearance.
 Descriptive rather than specific.
GLYCOGEN
 Seen in patients affected by enzymatic or storage disorders of
glucose metabolism.
 Diabetes Mellitus, glycogen is found on renal tubular cells, liver
cells, islet of Langerhans, cardiac myocytes.
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 Glycogen storage diseases or Glycogenoses have enzymatic
defects or storage defect.
PIGMENTS
Exogenous Pigments
 Common exogenous pigment is CARBON.
 Accumulation of this pigment blackens the tissue (Anthracosis).
 Coal Worker’s Pneumoconiosis.
 Tattooing is also a form of localized exogenous pigmentation
where in the ink is being phagocytosed by dermal macrophages.
Endogenous Pigments
Lipofuscin
 An insobluble pigmen, also known as lipo-chrome or wear-and-
tear pigment.
 Derived through lipid peroxidation of polyunsaturated lipids.
 Not injurious but an indication of cell injury and lipid
peroxidation.
 Appears as brown lipid or brown in actual slides.
Melanin
 Brown-black pigment.
 Clinically significant in PKU, deficient homogentisic acid oxidase
leading to alkaptonuria.
 Pigments deposit in the skin, connective tissue, and cartilage, a
condition known as ochronosis.
Hemosiderin
 Hgb-derived golden yellow to brown granular or crystalline
pigment.
 One of the major storage forms of Iron.
 In systemic or local excess iron, ferritin forms hemosiderin.
 Hemosiderin represents aggregates of ferritin micelles.
 Local excess example is bruise.
 Systemic overload of iron, hemosiderin can be deposited in
organs causing a condition called hemosiderosis, caused mainly
by:
 Increased absorption of dietary ion.
 Hemolytic anemia
 Repeated blood transfusion
PATHOLOGIC CALCIFICATION
 Abnormal tissue deposition of calcium salts together with small
amounts of other minerals.
 Can be divided into Dystrophic or Metastatic.
Dystrophic Calcification
 Occurs despite normal serum levels of calcium.
 Encountered in areas of necrosis, whether they are coagulative,
caseous, or liquefactive, also in fat necrosis.
 Calcium appears as fine, white granules or clumps often felt as
gritty deposits.
 Ex: Valvular Stenosis
Metastatic Calcification
 Occurs in tissue due to disturbances in calcium metabolism.
 Mineral salts ‘usually’ cause no clinical dysfunction.
 Can be cause by:

1.) Increased secretion of PTH

 Hyperparathyroidsim
 Increased bone resportion of Calcium

2.) Resorption of Bone Tissue

 Secondary to primary bone tumors.

3.) Vitamin-D related disorders

 Sarcoidosis, Idiopathic Hypercalcemia.

4.) Renal Failure

 Causes phosphate retention. DEFECTIVE PROTEIN HOMEOSTASIS
 Usually secondary to hyperparathyroidism.  Protein homeostasis is maintained by 2 mechanisms:
 Maintain in proper folding state
CELLULAR AGING  Degrade misfolded ones by autophagy-lysosome system.

DEREGULATED NUTRIENT SENSING

 Eating less, increases longevity.
 Caloric restriction increases life span.
 IGF-1 mimics insulin hormone, informing the cell the availability
of glucose promoting anabolic state.
 Sirtuins are family of NAD-dependent protein deacetylases that
are designed to adapt to bodily functions to various
environmental stresses, including food deprivation.

 Result of a progressive decrease in cell function and viability.

 Caused by:
 Genetic Abnormalities
 Accumulation of cellular and molecular damage.

DNA DAMAGE
 Defective DNA repair mechanisms.
 Accumulation of chromosomal damages.

CELLULAR SENSENCE
 Limited capacity for replication, after a fixed number of divisions,
the cell arrests in a terminally non-dividing state.
 Telemere Attrition – progressive shortening of telomeres result “Pyryzy napasirossa vokemilzi, va daorunta nelli
to cell cycle arrest. qringaomna nojo zalari”
 Activation of Tumor Suppresion Genes – CKN2A locus encode (They will purify nonbelievers by the thousands, burning
TSGs that help regulate cell senescence. their sins and flesh away)

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