Pathobiology Program
Cellular Injury and Cell
Death
Angelo M. De Marzo MD PhD
Aug. 25, 2014
Pathology
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Study of structural, biochemical, and
functional changes in cells, tissues and
organs that underlie disease.
Use of molecular, microbiologic,
immunologic, and morphologic techniques to
explain the signs and symptoms of disease
and provide a basis for treatment.
Serves as bridge between basic sciences
and clinical medicine (scientific foundation for
medicine)
Robbins and Cotran, Pathological Basis of Disease, 9th Edition
Case
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50 year old
male with
sudden left
sided
paralysis
Questions:
•How is this manifested at
the cellular and molecular
level?
•Can help with
developing new Rx
to reduce impact of
injury and inform us
about new cancer
Rx approaches
Robbins and Cotran, Pathological Basis of Disease, 7th Edition
Introduction to Pathology
Etiology or Cause of Disease
 The inciting agent (i.e. HIV virus infection)
Pathogenesis (How it happens)
 Sequence of events in response of cells or
tissues to the etiologic agent, from the initial
stimulus to the ultimate expression of the disease
(i.e. HIV infection  CD4+ T cell death 
opportunistic infection of lung  leading to
pulmonary failure  leading to cardiac failure 
death)
Pathology
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Anatomic pathology: study
changes in structure, or
appearance of organs, tissues
and cells as related to changes
in function
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Morgagni's Seats and causes of
disease ( 1761): symptoms
explained in terms of anatomical
changes observed at post-mortem
Functional Derangements and
Clinical Manifestations
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Organ injury starts with molecular or
structural alterations in cells (nineteenth
century by Rudolf Virchow)
The nature of the morphologic changes and
their distribution in different organs or tissues
alter function and determine the clinical
features (symptoms and signs), course, and
prognosis of the disease
Morphologic Changes
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Structural alterations in
cells or tissues that are
either characteristic of the
disease or diagnostic of
the process
Diagnostic pathology is
devoted to identifying the
nature and progression of
disease by studying
morphologic changes in
tissues
Modern Diagnostic Pathology
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Molecular analyses
augment anatomical
approaches:
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Mutational analysis
FISH
gene expression
microarrays
Immunohistochemistry
Uncovering changes or
“signatures” that bear
on the behavior of the
disease
Molecular Pathology
Her-2-neu amplification in
breast cancer
Robbins and Cotran, Pathological Basis of Disease, 9th Edition
Robbins and Cotran, Pathological Basis of Disease, 9th Edition
Robbins and Cotran, Pathological Basis of Disease, 9th Edition
Hypertrophy
Robbins and Cotran, Pathological Basis of Disease, 7th Edition
Atrophy
Shrinkage in the size of the
cell by loss of cell substance
 A form of adaptive response
 When many cells are involved, the entire tissue or
organ diminishes in size, or becomes “atrophic”
 Can be physiologic or pathologic
 The uterus decreases in size shortly after parturition
 Pathologic atrophy depends on the underlying cause
and can be local or generalized
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Robbins and Cotran, Pathological Basis of Disease, 9th Edition
Causes of Atrophy
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Decreased workload
(atrophy of disuse)
Loss of innervation
(denervation atrophy)
Diminished blood
supply
Inadequate nutrition
Loss of endocrine
stimulation
Aging (senile atrophy)
Pressure
Normal Breast Tissue
Non Lactating
Lactating
Metaplasia
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a reversible change in
which one adult cell
type (epithelial or
mesenchymal) is
replaced by another
adult cell type
Commonly seen prior to
cancer development –
e.g. cervix, esophagus,
stomach
Robbins and Cotran, Pathological Basis of Disease, 7th Edition
Causes of Cell Injury/Death
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Hypoxia/Ischemia – most common cause (e.g.
myocardial infarction)
Physical agents
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Trauma, temperature extremes, radiation, electric shock
Chemical agents – (e.g. acid damage to
esophagus)
Infectious agents
Immunological reactions (e.g. autoantibodies)
Genetic derangements (e.g. Duchenne muscular
dystrophy or sickle cell disease)
Nutritional imbalances – too little or too much
Mechanisms of Cell Injury/Death
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Plasma membrane damage
Mitochondrial damage
Ribosomal damage
Nuclear damage
Oxidative Stress Induced
Damage
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Free radicals can produce profound cellular injury
Produced by
 Powerful external energy source – e.g. ionizing radiation
 Oxidative-reductive reactions
 Inflammation
Unpaired electrons are free to participate in chemical bond
formation
 oxidative DNA damage –
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Sugar damage leading to strand breaks
Base damage
Lipid peroxidation – creating chain reaction
Protein Oxidation
Nitrosative Damage is also important
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© 2005 Elsevier
Reversible Cell Injury: e.g. Fatty Liver
Fatty Liver
Normal Liver
Reversible Injury - Kidney
Robbins and Cotran, Pathological Basis of Disease, 9th Edition
Cell Death
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Plays a central role in multi-cellular
organisms during their early development in
sculpting body parts and in adult life by
controlling cell numbers
Further protects the organism overall by
removal of cells damaged by disease, aging,
infection, genetic mutation and exposure to
toxic agents
Traditionally broken down into
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Necrosis
Apoptosis
Necrosis
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Results from a variety of accidental and lethal
actions by toxins or physical stimuli or in
association with pathological conditions such as
ischemia.
Characterized by cellular swelling, dissolution of
nuclear chromatin, disruption of the plasma
membrane, and release of intracellular contents
into the extracellular space, resulting in
inflammation.
Traditionally considered to be a passive process
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However, it is now known to be a genetically
controlled process, at least at times (Peter, ME, Nature 471:310-,2011)
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© 2005 Elsevier
Time Course of Cell Injury and
Necrosis
Robbins and Cotran, Pathological Basis of Disease, 7th Edition
Necrosis
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Necrosis: from “accidental”
death of cells and living
tissue
Less orderly than apoptosis
Lack cell signals which tell
nearby phagocytes to
engulf the dying cell
The release of intracellular
content after cellular
membrane damage is the
cause of inflammation in
necrosis
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Emerging evidence that
specific proteins may mediate
this (e.g. HMGB1 and HDGF)
Examples of
“necrosis” at
the gross
level
Robbins and Cotran, Pathological Basis of Disease, 7th Edition
More Examples of Grossly
Apparent “Necrosis”
Necrosis at the Light Microscopic Level Coagulative Necrosis – Renal Infarct
Normal Tubules
Necrotic Tubules
In Coagulative necrosis, the outline of the original cells remain intact, but
nuceli are essentially dissolved
Coagulative Necrosis – Myocardial
Infarct
Normal Myocardium
Necrotic Myocardium
Another “type” of Necrosis: Liquifactive
Necrosis – Abscess – Microscopic
Appearance
In liquifactive necrosis, the outline of the original cells does not remain intact,
all local tissue is dissolved
Other Types of Necrosis Distinctly
Visible Grossly and Microscopically
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Fat Necrosis
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In some tissues (i.e. pancreas),
when cells die, lipases are
released  lysis of fat in
adipocytes in surrounding tissues
into free fatty acids  Ca+ salts
of these form “soap” deposits,
which are chalky white
Caseous Necrosis
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A specific type of liquifactive
necrosis occurring in
“granulomatous” inflammation
(i.e. tuberculosis)
http://www.sciencephoto.com/media/260971/enlarge
http://quizlet.com/3021100/pathology-slides-flash-cards/
Casesous Necrosis – Microscopic
Appearance (Lung)
Fibrinoid necrosis
Apoptosis (first recognized form
of programmed cell death)
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First evidence of a genetic program that
orchestrates physiological cell death came from
developmental studies of the nematode
Caenorhabditis elegans.
Knowledge increased dramatically in last 3 decades
with discovery of several death genes in C. elegans
and counterparts in mammals.
Dysregulation of apoptosis occurs in a wide variety
of diseases including cancer, autoimmune diseases,
neurodegenerative diseases, ischemic disease and
viral infections.
Apoptosis
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Coined by Kerr, Wyllie, and Currie
(Brit J. Cancer 26:239) in 1972
a morphologically distinctive form of
cell death that can be associated
with normal physiology
distinguished from necrosis
characterized by nuclear chromatin
condensation, cytoplasmic shrinking,
dilated endoplasmic reticulum, and
membrane blebbing.
http://www.nih.gov/sigs/aig/Aboutapo.html
Apoptosis
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Triggered by a wide variety of stimuli and occurs
normally in some organs during development
DNA damage (by irradiation or drugs)
Some hormones such as corticosteroids lead to death
in particular cells (e.g., thymocytes)
Sometime triggered by removal of survival factors (i.e.
androgens and prostate)
Apoptosis – Scanning EM
http://fig.cox.miami.edu/~cmallery/255/255hist/mcb1.19.apoptosis.jpg
Apoptosis – Transmission EM
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© 2005 Elsevier
Apoptosis – Transmission EM
http://www.msu.ac.th/bio-dept/Apoptosis.htm
Nature Reviews Mol
Cell Bio; 9:231-, 2008
Apoptosis – Hematoxylin &
Eosin – in human tonsil tissue
Apoptotic germinal center B cells are found within the
cytoplasm of macrophages= “tingible body macrophages”
http://library.med.utah.edu/WebPath/HEMEHTML/HEME121.html
Classical Forms of Cell Death
Necrosis
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Stimuli include toxins, severe
hypoxia, massive insult, and
conditions of ATP depletion.
There is no energy requirement.
It usually affects large numbers of
contiguous cells such as death of
patches of tissue.
Cell swells; organelles swell;
disruption of organelles occurs.
DNA breakdown pattern gives
randomly sized fragments.
See smear pattern in gel
electrophoresis.
Plasma membrane is lysed.
Inflammation occurs.
Chemotactic factors stimulate
neutrophil infiltration to degrade
dead cells.
Until recently was assumed to be
entirely passive!
http://www.msu.ac.th/bio-dept/Apoptosis.htm
Apoptosis
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Stimuli include physiological and
pathological conditions without ATP
depletion.
It is ATP dependent.
It usually affects scattered individual
cells such as death of single
isolated cells.
Cell contracts; chromatin
condenses; apoptotic bodies form.
DNA breakdown pattern gives DNA
fragments in multiples of about 200
base pair units. See ladder pattern
in gel.
Plasma membrane remains intact
and blebbed.
No inflammation occurs.
Apoptotic bodies are phagocytized
and intact; generally no neutrophil
infiltration occurs.
Nature Reviews Mol Cell Bio; 9:231-, 2008
T cells to directly kill infected cells
Death receptor agonists
(e.g. to kill self reactive T cells in thymus)
DISC= death-inducing
Signaling complex
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DNA Damage,
Lack of growth factor
signaling
Protein misfolding
Nature Reviews Mol Cell Bio; 9:231-, 2008
Nature Reviews Molecular Cell Biology 9, 378390 (May 2008)
Classical DNA Ladder of
Apoptosis
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© 2005 Elsevier
Nature Reviews Mol Cell Bio; 9:231-, 2008
Nature Reviews Mol Cell Bio; 9:231-, 2008
Nature Reviews Mol Cell Bio; 9:231-, 2008
Nature Reviews Mol Cell Bio; 9:231-, 2008
Apoptosis Movie
Why Apoptosis vs Necrosis?
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Necrosis -> rapid loss of membrane integrity
and release of cellular contents that trigger
inflammation > danger signals!
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Danger Associated Molecular Patterns stimulate
pattern recognition receptors on innate
inflammatory cells that then can activate adaptive
immunity
This makes sense since pathogens can provoke
necrosis, and, trauma which causes necrosis
promotes pathogen growth…
Yet, this can also result in “autoimmune”
recognition if not controlled
Potential Problems Solved by
Apoptosis
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The membrane changes (i.e. flip phosphatidyl
serine) facilitate removal of the cells by
phagocytes before contents spill
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Many cellular contents are protected from immune
system recognition
Apoptosis “shreds the evidence” so these
contents (i.e. chromatin, nuclear proteins like
fibrillarin) cannot elicit immune response =>
prevents the “unmasking of hidden self”
Autophagy
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Sequestration of cytoplasm and organelles by
double or multi-membrane structures called
autophagic vacuoles, followed by degradation
of the contents of the vacuoles by fusing to
lysosomes
Long regarded as a cell survival mechanism:
e.g. may help cancer cell survive under
nutrient-limiting conditions
If carried to exhaustion, digestion of own
components results in cell death
Autophagy
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Electron microscopy observations of the
appearance of double membrane–enclosed
structures termed autophagosomes
Microtubule-associated protein 1 light chain 3
(LC3) is the homolog of yeast Atg8, an
autophagy-related protein
Autophagy Detection in Cell
Culture
Programmed
Necrosis/Necroptosis
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Support as “non-passive” process came from
studies of death receptors
Activate FAS or TNFR, prototypic extrinsic
apoptosis induction (recruit FADD and
upstream caspase 8)
But… in certain cell types same stimulation in
a “apoptosis deficient condition”, produces
cell death with morphology of necrosis
“The fact that the activation of Fas/TNFa receptors may lead to cell death with
features of either apoptosis or necrosis argues strongly for the existence of a
regulated cellular necrosis mechanism, discrete from apoptosis, which we termed
‘necroptosis’’
Hitomi, et al., 2008, Cell 135, 1311-1323
Necroptosis
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RIP1 = kinase associated with death
receptors but kinase activity not needed for
apoptosis
Block apoptosis and RIP1 kinase activity is
needed for activation of necroptosis by death
receptor agonists
Nec-1 = necrostatin, a small molecule
inhibitor of necroptosis, is allosteric inhibitor
of RIP1 Kinase (tool to determine if
necroptosis is involved)
Apoptosis
FAS + L929
Cells
+ Z.VAD.fmk
(caspase inhibitor)
Necroptosis
+Z.VAD.fmk
+ Nec-1
Little cell death
432 genes involved in necroptosis, 32 are downstream or
regulators of RIP1, 7 genes involved in both necroptosis and
apposes, including Bmf (BH3-only protein)
Hitomi, et al., 2008, Cell 135, 1311-1323
Mitochondrial Permeability
Transition Pore
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Non-selective pore for which the
precise structural identity is not well
defined
Nonselective water and solutepassing protein channel spanning the
inner and outer mito membranes
Closed in unstressed cells, and open
upon oxidative stress, causing
massive ion influx that dissipates the
membrane potential and shuts down
ATP production
Regulated by cyclophilin D (a propyl
isomerase)
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CypD-/- mice are resistant to ischemia
induced necrosis in MI and stroke and
CypD deficient mito and cells are resistant
to Ca2+ and H202 induced cell death, but
are sensitive to Bcl-2 family driven
apoptosis
Cell Death and Differentiation (2009) 16, 1419–1425
Programmed Necrosis
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Molecular Players
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Mediated by the opening of mitochondrial permeability transition
pores (MPTP), which disrupts mitochondrial respiratory function
Depends on RIP1 and RIP3 Kinases (Cho et al., Cell, 137:11121123, 2009)
Cyclophilin D (CypD) (Halestrap, Biochemical Society Transactions
34, 232–237 2006)
Newer data implicating p53 (Vaseva A, Cell,149:1536-1548,
2012) as well as ALKBH7 (Genes Dev. 27: 1089-1100, 2013)
Regulates disease pathologies in animal models of
hypoxic/ischemic injury acute pancreatitis and septic shock
Why Programmed Necrosis
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May act as a second line of defense in viral
infections.
Viruses may encode caspase inhibitors to
block apoptosis
Viral inhibitor of RIP1 has been founds (M45)
RIP3 -/- cells are protected against viral
induced cell death
Thus, necroptosis may function to stimulate
the immune system in response to infection
Cross talk between Apoptosis
and Necroptosis
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In development, activation of apoptotic pathways results in
repression of necrotic pathway
Nature 471: 311-, 2911.
Crosstalk between Autophagy
and Necroptosis
Treatment of RCC with PI3K inhibitors results
in induction of autophagy and suppression of
necroptosis by a reduction in RIPKs.
 Simultaneous treatment of RCC with PI3K
inhibitor and autophagy inhibitor results in
induction of RIPK and ROS-mediated
necroptosis
Thus, autophagy is a survival mechanism to
RIPK-dependent necroptosis in the setting of
cancer therapy
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PLoS ONE: 7:7, e41831, 2012
There Can Be Too Much and Too
Little Programmed Cell Death
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Too much:
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AIDS
ischemia and cardiac and stroke pathology
neurodegenerative diseases
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Parkinson’s, Huntingtons, ALS
Too little: Many cancers
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P53 is activated in response to DNA damage
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Cells having excessive DNA damage can develop
mutations and should die, but tumor cells can develop
blocks in apoptotic pathways (i.e. by mutating p53) or
perhaps programmed necrosis
Questions?