Cellular Adaptation and Cell Injury
CLDavis Foundation
On the Beach
Lecture 2
Hypoxia,
yp
, reperfusion,
p
, and apoptosis
p p
R K Myers 2009
Hypoxic cell injury
Hypoxia: any state of reduction of O2 supplied to cells and tissues
and results in decreased ATP production.
Hypoxia can result from:
cardiorespiratory failure
loss of blood supply
reduced transport
p of O2 in blood ((anemia or CO toxicosis))
blockage of cell enzymes (e.g. cyanide)
Ischemia is decreased blood supply or perfusion of tissues usually
due to constriction or obstruction of blood vessels.
Ischemia tends to injure tissue faster because substrates for
glycolysis are not delivered, anaerobic generation of ATP stops
faster, glycolytic function is inhibited by metabolite accumulation.
Consequences of hypoxia depend
on cell type
Highly vulnerable to hypoxia:
Relatively resistant to hypoxia:
cardiac myocytes
Adipocytes
neurons
Bones
proximal tubule epithelium
p
p
Skin
endothelial cells
Muscle
pneumocytes
Significance of hypoxia depends on:
Organ/Cell type
Degree
Duration
Extent
1
Events in ischemia: reversible and
irreversible injury
Ischemia -- Reperfusion Injury
• When blood flow is restored to previously
ischemic but not dead cells, injury may
become worse and be accelerated.
• Loss of cells in addition to cells that are
irreversibly damaged at the end of
ischemia.
• Very important clinically in myocardial
infarction and stroke.
Ischemia -- Reperfusion Injury
• Reoxygenation followed by increased generation
of free radicals
• Compromised cellular antioxidant defenses
• Promotion of the mitochondrial permeability
transitionÆ precludes mitochondrial energization
and ATP recovery
• Inflammation resulting from cytokines and
increased adhesion molecules from
parenchymal and endothelial cells. Leukocyte
influxes add to damage.
• Activation of complement pathway
2
Reperfusion injury
• Exuberant free radical formation occurs
with reperfusion of parenchymal,
endothelial cells, and leukocytes due to:
– Xanthine oxidase pathway
– Mitochondrial electron transport chain
– Conversion of NOS to produce superoxide
(rather than NO)
– NADPH oxidase from infiltrating leukocytes
Mark Ackermann, Iowa State U. 2008
Free radical formation during
reperfusion: xanthine oxidase
Cell ischemia/loss of oxygen leads to ATP degradation. If perfusion is
re-established, oxidative radicals are formed.
ATP
ISCHEMIA
ADP
Xanthine dehydrogenase
AMP
Adenosine
Inosine
Xanthine oxidase
O2
-
O2
Xanthine oxidase
Xanthine
Hypoxanthine
SOD
Uric acid
H2O2
Fe++
REPERFUSION
.
Tissue injury
OH
Mark Ackermann, Iowa State U. 2008
Proximity of muscle and other cells to
endothelial cells in reperfusion injury
• Direct contact between myofibers and other cells
to endothelial cells allows passage of
nucleotides via nucleotide transport proteins
(NTP) that can be used in the xanthine oxidase
pathway
th
Muscle or other cell
ATP-ADP-AMP-Adenosine-Inosine
NTP
NTP
Adenosine-Inosine
X.O. pathway
Endothelial cell
Mark Ackermann, Iowa State U. 2008
3
Mitochondrial activity contribution to ROS in
reperfusion injury
• Distal to NADPH dehydrogenase in the
mitochondrial electron transport chain
– Ubiquinone (CoQ) increases ROS
formation
• ROS induce mitochondrial permeability
transition (MPT)
• These contribute to ROS formation and
reperfusion injury
Mark Ackermann, Iowa State U. 2008
Increased superoxide generation from NOS
during reperfusion injury
• All three NOS (nNOS, eNOS and iNOS) can
switch from NO to superoxide generation
– This occurs with depletion of NOS substrate (arginine
and BH4)
• Both arginine and BH4 are depleted by ROS (vicious cycle)
• The increased superoxide production
contributes to ROS damage and reperfusion
injury
Mark Ackermann, Iowa State U. 2008
Leukocyte infiltration with reperfusion injury
• Hypoxic tissues, including myocytes and
endothelial cells increase expression of
leukocyte adhesion molecules
– Enhanced leukocyte (neutrophil) infiltration
with reperfusion
– Increased activity of NADPH oxidase by
infiltrating leukocytes
• NADPH oxidase produces additional ROS
Mark Ackermann, Iowa State U. 2008
4
Reperfusion injury therapy
• Therapies
– Needed to reduce ROS damage but yet
allowing perfusion and return to normoxia
– Antioxidants
– Anti-neutrophil
Cell Death by Apoptosis
• Apoptosis (apoptotic necrosis) a type of
programmed cell death with initiation of a selfinduced cell death process (“cell suicide”)
• A variety
i t off stimuli
ti li resultlt in
i self-programmed,
lf
d
genetically determined, energy-dependent
sequences of molecular events involving
initiation by cell signaling, control and integration
by regulatory molecules, a common execution
phase by caspase family genes, and dead cell
removal
Cell Death by Apoptosis
• Initiation phase: caspases (cysteine
proteases that cleave aspartic acid
residues) become catalytically active by
intrinsic and extrinsic paths
paths.
• Execution phase: specific caspase
enzymes act to cause cell death
5
Buds
(or blebs)
Blebs
The sequential ultrastructural changes seen in necrosis (left) and apoptosis
(right). In apoptosis, the initial changes consist of nuclear chromatin condensation and
fragmentation, followed by cytoplasmic budding and phagocytosis of the extruded
apoptotic bodies. Signs of cytoplasmic blebs, and digestion and leakage of cellular
components characterize necrosis. (Adapted from Walker NI, Harmon BV, Gobe GC, Kerr JF: Methods Achiev
Exp Pathol 13:18-54, 1988.)(McGavin, M. Donald. Pathologic Basis of Veterinary Disease, 4th Edition. C.V. Mosby,
Morphologic features of
oncotic and apoptotic cell
death
Oncotic Necrosis Morphology:
•.Groups of cells swell and then
may shrink
•.pyknosis Æ karyorrhexis Æ
karyolysis
•.Cytoplasm is disrupted
•.Cytoplasmic blebs form.
Enzymatic digestion, contents may
leak from cell
•.Inflammation is frequent
•Invariably pathologic
Apoptotic Cell Death Morphology:
•.Individual cells are shrunken.
•.Chromatin is condensed. Fragmentation
into nucleosome size fragments (pyknosis
and karyorrhexis)
•.Cytoplasm is fragmented, membranes
intact
•.Cytoplasmic buds form and may be
found in adjacent cells and phagocytes as
apoptotic bodies.
•.Inflammation is absent
•Can be physiologic or pathologic
Causes of Apoptosis
• Normal occurrence in many situations,
physiologic and pathologic
• Eliminates unwanted, potentially harmful,
useless, damaged cells.
• DNA damage of many types is a cause,
e.g. due to failure of DNA repair
6
Physiologic Apoptosis
• Programmed cell destruction during
embryogenesis (implantation, organogenesis,
developmental involution, metamorphosis. PCD
• Hormone dependent involution in adults. E.g.
endometrial and uterine involution
• Cell
C ll d
deletion
l ti iin proliferating
lif ti cellll populations.
l ti
Skin (see later), gut.
• Death of inflammatory and immune cells
• Elimination of self reactive lymphocytes
• Cell death by cytotoxic T cells. Defense
mechanism against viruses, tumors, transplants
Pathologic Apoptosis
• Cell death produced by injurious stimuli.
– Radiation, anticancer drugs that damage
DNA, heat, hypoxia (if mild), ER stress
induced by unfolded proteins
• Cell injury
j y in some viral diseases.
• Pathologic atrophy in parenchymal organs
after duct obstruction.
• Cell death in tumors
• Can be seen with or precede oncotic
necrosis
Dysregulated Apoptosis
(too little or too much)
May be important in a wide range of diseases
• Disorders associated with defective apoptosis and
increased cell survival
– Cancers. Tumors with p53 mutations common. Also hormonedependent tumors (mammary, prostate, ovary)
– Autoimmune disease
disease. May arise from failure to eliminate auto
reactive lymphocytes after encounter with self antigens.
• Disorders associated with increased apoptosis and
excessive cell death. Excess loss of normal or
protective cells.
– Neurodegenerative diseases with loss of specific neuron subsets
– Ischemic injury of myocardial infarcts and stroke
– Death of virally infected cells
7
Biochemical Features of Apoptosis
• Protein cleavage: protein hydrolysis and activation of
caspase family proteases (at least 13 known). Present
normally as inactive pro-enzymes that need to be
activated. Results in degradation of nuclear scaffold and
cytoskeleton
• DNA breakdown. Activation of DNAases. Breakdown to
50-300 kilobase pieces with subsequent cleavage of
DNA into nucleosomes in multiples of 180-200 base
pairs (“ DNA ladders”)
• Phagocytic recognition. Phosphatidylserine “flipped” out
from inner layer to outer layer permitting recognition by
macrophages (“eat me”) without inflammation.
Mechanisms of Apoptosis
Mechanisms of Apoptosis
Initiation phase. Signals from 2 (or more) distinct
but convergent pathways.
• Extrinsic (death receptor-initiated) pathway
• Receptor-ligand
p
g
interactions:
– TNF receptor (TNFR1). Tumor necrosis factor family
– Fas – Fas ligand.
• Active caspase 8 leads to executioner path
• Can be inhibited by FLIP – used by some
viruses to protect virally infected cells from Fas
apoptosis
8
Extrinsic (death receptorinitiated) pathway of
apoptosis
1. Fas cross linked to
FasL.
2. 3 death domains (FAD)
come together and form
binding site for an
adaptor protein.
3 FADD attached to death
3.
receptors binds inactive
caspase-8 which come
together and
autocatalytically activate
to active caspase-8
4. Cascade of other
caspase activation
activates executioner
caspases.
5. Apoptosis initiated
Mechanisms of Apoptosis
Intrinsic (mitochondrial) pathway: in general
• Result of increased mitochondrial permeability and
release of pro-apoptotic molecules into cytoplasm (no
death receptors)
• Mitochondrial outer membrane permeabilization (MOMP)
leads to release of proteins from mitochondrial
intermembrane space into the cytosol
• Initiated by many types of injury: radiation, toxins, free
radicals, hypoxia, withdrawal of growth factors or
hormones (which stimulate production of Bcl-2 (B cell
lymphoma) family anti-apoptotic components (especially
Bcl-2 and Bcl-x) that reside in mitochondrial membranes.
• Can be inhibited by FLIP – used by some viruses to
protect virally infected cells from Fas apoptosis
The intrinsic (mitochondrial) pathway of apoptosis. Death
agonists cause changes in the inner mitochondrial membrane,
resulting in the mitochondrial permeability transition (MPT) and
release of cytochrome c and other pro apoptotic proteins into the
cytosol, which activate caspases. AIF, Apoptosis-inducing factor.
From Kumar V, Abbas A, Fausto N: Robbins & Cotran pathologic basis of disease, ed 7, Philadelphia, 2005,
Saunders.)(McGavin, M. Donald. Pathologic Basis of Veterinary Disease, 4th Edition. C.V. Mosby,
9
Mechanisms of Apoptosis
Mitochondrial disruption: two killing pores
•
Outer-membrane permeability
– MOMP: Mitochondrial outer membrane permeabilization
– Cytochrome c from the mitochondrial intermembrane space initiates
caspase activation and apoptosis
•
Loss of outer membrane integrity results in release of several killing
molecules
– Cytochrome c, apoptosis-inducing factor (AIP), Smac/Diablo,
Omi/HtrA2, et al.
•
Triggers of MOMP mitochondrial pathway:
– Anything contributing to cellular stress or loss of housekeeping
– Nutrient deprivation, unfolded proteins, cytoskeletal disruption, DNA
damage, ion imbalance, toxins
– Developmental signals: cytokines, steroids, lipid mediators,
immunologic effector processes
Mechanisms of Apoptosis
Mitochondrial disruption: two killing pores
•
Outer-membrane permeability
•
Two models for how outer membrane becomes permeable to
intermembrane proteins
•
MOMP without decreased inner membrane integrity may be mediated by
Bcl 2 proteins Bax and Bak (pro
Bcl-2
(pro-apoptotic
apoptotic with 3 Bcl-2
Bcl 2 homology – BH–
BH
domains). Model 1
– These oligomerize in the outer membrane and with membrane lipids form pores
to leak intermembrane proteins
– (pro-apoptotic one BH domain Bcl-2 family members, e.g. Bid, Bad, Bim, etc.,
promote pores by activating Bax/Bak
– Anti-apoptotic Bcl-2 members (Bcl-2 and Bcl-XL) antagonize the process
•
Model 2. MOMP is a direct effect of MPT (mitochondrial permeability
transition), which is a pore defect of the inner membrane. This assumes
mitochondrial swelling, which is not necessary for apoptosis.
Mechanisms of Apoptosis
Mitochondrial disruption: two killing pores
• Mitochondrial permeability transition (MPT)
– Results from pore development in inner membrane—transient in
physiological stress but permanent under injury conditions,
including Ca overload
– MPT pores distinct from pores leaking intermembrane proteins
– Inner p
pore may
y contact outer membrane
• Proteins implicated in MPT
– ANT = adenine nucleotide translocase
– VDAC = voltage-dependent anion channel
– Cyclophilin D (inhibited by cyclosporin A which retards MPT)
• MPT promoted by:
– Ca overload
- Oxidative stress
– Phospholipid hydrolysis - ATP depletion
– Diminished mitochondrial membrane potential
10
Mechanisms of Apoptosis
Perforin/Granzyme Pathway
• Cytotoxic T cells. CD8+ cells kill antigen-bearing cells.
A variant of type IV hypersensitivity.
• A novel pathway in addition to extrinsic and FasL/FasR
predominantly
y used by
y CTL-induced
interaction p
apoptosis.
• Perforin, a transmembrane pore forming molecule is
followed by exophytic release of cytoplasmic granules
through the pore into the target cell. Granzyme A and B
(serine proteases) are the major component of the
granules.
Mechanisms of Apoptosis
Perforin/Granzyme Pathway
• Granzyme B cleaves proteins at aspartate residues and
activates pro-caspase 10
• Granzyme B can also use mitochondrial path to amplify
the death signal
g
by
y release of cytochrome
y
c. It can also
directly activate caspase 3.
• Granzyme A activates caspase independent paths,
activating DNA nicking via DNAase NM23-H1, a tumor
suppressor gene product.
• Effects on virally infected cells, tumor cells, and immune
modulation
Mechanisms of Apoptosis
Execution Phase
• Proteolytic cascade at convergence of other
paths
• Executioner caspases are caspase 3 and 6
• Pro-enzymes are activated by other caspases
(e.g. 8 and 9) or autocatalytically
• Executioner caspases: cleave cytoskeletal and
nuclear matrix proteins, disrupting cytoskeleton
and breaking down nucleus.
11
Mechanisms of Apoptosis
Execution Phase
• Nuclear targets of caspase activation;
–
–
–
–
Transcription proteins
DNA replication
DNA repair
Conversion of cytoplasmic DNAase into active form
by caspase 3 cleaving of inhibitor of the enzyme.
• Effect is induction of Internucleosomal cleavage
of DNA
Mechanisms of Apoptosis
Removal of dead cells.
• Phospholipid asymmetry and externalization of
phosphatidylserine on the cell surface (flip) is hallmark.
• Surface phosphatidylserine and other molecules recruit
phagocytes leading to noninflammatory phagocytic (and
adjacent cell) recognition (early uptake and disposal
without inflammation)
• Many macrophage receptors are involved in binding and
engulfing apoptotic cells.
• Macrophages secrete substances that bind to apoptotic
but not live cells resulting in opsonization for
phagocytosis.
• Viable cells prevent engulfment by macrophages by
expression of surface molecules (CD31)
• The result: no inflammation. Disappearance without a
trace.
Mechanisms of Apoptosis
Summary
Initiators of apoptosis: TNF, nitric oxide, fas ligand, granzyme, viral infection,
radiation, corticosteroids, DNA damage
Inhibition of apoptosis: Growth factors, differentiation factors, adequate
intracellular nutrition, insulin, others
Extrinsic signaling
– Ligand (TNF, CD95L (Fas Ligand), Trail
– Activates
A ti t Pro-caspase
P
8 tto caspase 8 ((active)
ti )
Intrinsic signaling
– DNA damage (p53), UV damage, viral infection, cell injury
-- Cytochrome c release and binds to Apaf-1 to activate caspase 9, Bcl-2
and Bcl-x (anti apoptotic) lost and replaced by Bax, Bak, etc.
Initiator caspases
• 2, 8, 9, 10, 12
Effector caspases:
• 3, 6, 7
12
Morphology of Apoptosis
Epidermal apoptosis with hypereosinophilic cytoplasm and small dense
nucleus. Right. Apoptotic liver cell
Apoptosis, cytoarchitecture of cells, pancreas, rat. Individual acinar cells are
shrunken and their chromatin condensed and fragmented (arrows). Cytoplasmic
buds are found in adjacent cells. Inflammation is absent. H&E stain. (Courtesy Dr.
M.A. Wallig, College of Veterinary Medicine, University of Illinois.)
(McGavin, M. Donald. Pathologic Basis of Veterinary Disease, 4th Edition. C.V. Mosby
Necrosis and apoptosis, mouse hepatitis virus infection, liver, mouse.
This disease causes hepatocyte death, typically by oncotic necrosis but
sometimes by apoptosis. Note areas of coagulation necrosis in the lower
left and apoptotic bodies in the center, some of which have been taken up
by adjacent hepatocytes (arrows). H&E stain.(McGavin, M. Donald. Pathologic Basis of
Veterinary Disease, 4th Edition. C.V. Mosby,
13
Assays for Apoptosis
Because oncotic and apoptotic necrosis
overlap, need to use 2 or more assays to
confirm death by apoptosis, one for
initiation phase and one for later execution
phase.
U d t d ki
Understand
kinetics
ti off cellll d
death
th and
d realize
li
apoptosis may be initiated and completed
within 2-3 hours. False negatives can
occur: too early or too late
Many assays (6 major groups) are available
with various strengths and weaknesses
Assays for Apoptosis
1.
Cytomorphologic alterations
H&E visualization. Misses early stages
Semi-ultrathin epoxy-resin blocks stained with Toluidine blue
TEM. Gold standard to confirm apoptosis.
Ultrastructural morphology.
•
•
•
•
•
•
•
•
electron dense nucleus (marginalized early)
nuclear fragmentation
intact cell membrane, even during disintegration
disorganized cytoplasmic organelles
large clear vacuoles
blebs (buds) on cell surface
loss of cell to cell adhesions
apoptotic bodies have intact cell membranes and cytoplasmic
organelles, with or without nuclear fragments.
Ultrastructure of apoptosis. Nuclear fragments with peripheral
crescents of compact chromatin or uniformly dense fragments.
14
Assays for Apoptosis
2. DNA Fragmentation.
1. Laddering Technique. Endonuclease
cleavage product on agarose gel.
2. TUNEL (Terminal dUTP Nick End-Labeling).
Assays endonuclease cleavage products by
enzymatically labeling DNA strand breaks.
Detection by light or fluorescence
microscopy or flow cytometry. Sensitive and
fast, but false positives from oncotic cells
and cells in DNA repair and gene
transcription.
Agarose gel
electrophoresis for
apoptosis.
A = control
B = culture of heated
cells showing
apoptosis. Note
‘laddering’
g of DNA
showing
nucleosome-sized
fragments (multiples
of 180-200 base
pairs).
C = culture of cells
with massive
necrosis. Note
smearing of DNA
Assays for Apoptosis
3. Detection of caspases, cleaved
substrates, regulators, and inhibitors
a. 13 known caspases detected by various
caspase activity assays and
immunohistochemistry.
b. Caspase activation detected by western
blot, immunoprecipitation, and IHC
c. Apoptosis PCR microarray. Uses real time
PCR to profile expression of up to 112
genes involved in apoptosis
15
Assays for Apoptosis
4. Membrane alterations
a. Phosphatidylserine externalization on outer
plasma membrane detected by Annexin V.
Tissues, embryos, or cultured cells.
b. Fluorescence microscopy, sensitive, but
oncotic necrotic cells also labeled.
c. Use dyes to mark oncotic cells
Assays for Apoptosis
5. Detection of apoptosis in whole mounts
6. Mitochondrial assays
a. Assays of cytochrome c release allows detection of
early phase of intrinsic path
b. Uses laser scanning confocal microscopy
c. Can also assess mitochondrial permeability
transition (MPT), calcium fluxes, mitochondrial
redox, status, and reactive oxygen species
d. Others including detection of apoptotic or antiapoptotic proteins (Bax, Bid, and Bcl-2) by
fluorescence and confocal microscopy.
Comparison of Necrosis and Apoptosis*
Apoptosis
Necrosis
•
•
•
•
•
•
•
•
•
•
•
•
Accidental cell death
Contiguous regions of cells
Cell swelling
Plasmalemmal bleb w/o
organelles
Small chromatin aggregates
Random DNA degradation
Cell lysis with release of cellular
components
Inflammation and scarring
Mitochondrial swelling and
dysfunction
Phospholipase and protease
activation
ATP depletion and metabolic
disruption
Cell death precipitated by plasma
membrane rupture
•
•
•
•
•
•
•
•
•
•
•
•
Controlled cell deletion
Single cell separating from
neighbors
Cell shrinkage
Zeiotic blebs containing large
organelles
Nuclear condensation and
lobulation
Internucleosomal DNA
degradation
Fragmentation into apoptotic
bodies
Absence of inflammation and
scarring
Mitochondrial permeabilization
Caspase activation
ATP and protein synthesis
sustained
Intact plasma membrane
*Molecular mechanisms of Cell Death. Molecular Pathology, Coleman and Tsongalis, 2009
16
Other forms of programmed cell death
besides apoptosis
• Cell death a has diverse array of phenotypes
• Other types of cell death may require gene activation
and energy dependence.
• Some have features of both oncotic and apoptotic
necrosis Æ ‘aponecrosis’
p
• Autophagy may represent another mechanism of PCD
Autophagic cell death has sequestration of cytoplasm
and organelles in double or multimembrane vesicles and
delivery to the cell’s own lysosomes for degradation.
Cannibalizes. Depends on protein synthesis and ATP.
Apoptosis: a review of programmed cell death. Elmore S.
Toxicol Pathol. 2007;35(4):495-516. Review.
Classification of Cell Death:
Recommendations of the Nomenclature
Committee on Cell Death (NCCD) 2009
Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, Blagosklonny MV, El-Deiry WS,
Golstein P, Green DR, Hengartner M, Knight RA, Kumar S, Lipton SA, Malorni W, Nuñez G, Peter ME, Tschopp J,
Yuan J, Piacentini M, Zhivotovsky B, Melino G; Nomenclature Committee on Cell Death 2009.
Cell Death Differ. 2009 Jan;16(1):3-11. Epub 2008 Oct 10
• Different types of cell death are defined by
morphological criteria without precise
biochemical mechanisms
• Proposes unified criteria for the definition of cell
death and of its different morphologies
• Definitions for cell death-related terminology
such as: ‘autophagic cell death’, ‘entosis’, mitotic
catastrophe’, necrosis, ‘necroptosis’, ‘pyroptosis’
When is a Cell Dead (NCCD)?
•
There is not a clearly defined biochemical event that
can be considered as the point-of-no-return
Cell should be considered dead when any one of the following
molecular or morphological criteria is met:
(1) Th
The cellll h
has llostt th
the iintegrity
t it off itits plasma
l
membrane,
b
as d
defined
fi d b
by th
the
incorporation of vital dyes (e.g., PI) in vitro.
(2) The cell, including its nucleus, has undergone complete fragmentation
into discrete bodies (which are frequently referred to as ‘apoptotic bodies’)
and/or
(3) Its corpse (or its fragments) has been engulfed by an adjacent cell in
vivo.
17
Table 2. Distinct modalities of cell death
Cell death mode
Apoptosis
Morphological features
•Rounding-up of the cell
•Retraction of pseudopods
•Reduction of cellular and nuclear volume (pyknosis)
•Nuclear fragmentation (karyorrhexis)
•Minor modification of cytoplasmic organelles
•Plasma membrane blebbing
•Engulfment by resident phagocytes
phagocytes, in vivo
Notes
‘Apoptosis’ is the original term introduced by Kerr et
al.14 to define a type of cell death with specific
morphological
features. Apoptosis is NOT a synonym of programmed
cell death or caspase activation. Subtypes of apoptosis
exist with heterogeneous functional aspects.
Categorization of cell death types. G Kroemer et al
Table 2. Distinct modalities of cell death
Cell death mode
Necrosis
Morphological features
•Cytoplasmic swelling (oncosis)
•Rupture of plasma membrane
•Swelling of cytoplasmic organelles
•Moderate chromatin condensation
Notes
‘Necrosis’ identifies, in a negative
fashion, cell death lacking the features
of apoptosis or autophagy. Note that
necrosis can occur in a regulated
fashion, involving a precise sequence of
signals.
Categorization of cell death types. G Kroemer et al
Table 2. Distinct modalities of cell death
Cell death mode
Autophagy
Morphological features
•Lack of chromatin condensation
•Massive vacuolization of the cytoplasm
•Accumulation of (double-membraned)
autophagic vacuoles
•Little or no uptake by phagocytic cells, in vivo
Notes
‘Autophagic cell death’ defines cell
death occurring with autophagy (but not
BY autophagy), though it may
misleadingly suggest a form of death
occurring by autophagy (this process
often promotes cell survival).
Categorization of cell death types. G Kroemer et al
18
Table 2. Distinct modalities of cell death
Cell death mode
Cornification
Morphological features
•Elimination of cytosolic organelles
•Modifications of plasma membrane
•Accumulation of lipids in F and L granules
•Extrusion of lipids in the extracellular space
•Desquamation (loss of corneocytes) by protease
activation
Notes
‘Cornified envelope’ formation or
‘keratinization’ is specific of the skin to create
a barrier function. Although apoptosis can be
induced by injury in the basal epidermal layer
(e.g., UV irradiation), cornification is exclusive
of the upper layers
(granular layer and stratum corneum).
Categorization of cell death types. G Kroemer et al
NCCD tentative definitions of Atypical
Cell Death Modalities: Examples
“Mitotic Catastrophe”
•
•
Cell death mode occurring during or shortly after a
dysregulated/failed mitosis
Can be accompanied by morphological alterations including:
– Micronucleation (which often results from chromosomes and/or
chromosome fragments that have not been distributed evenly between
daughter nuclei) and
– Multinucleation (the presence of two or more nuclei with similar or
heterogeneous sizes, deriving from a deficient separation during
cytokinesis).
•
•
•
No broad consensus on the use of this term.
Mitotic catastrophe can lead either to an apoptotic morphology or to
necrosis.
NCDD recommends the use of expressions such as 'cell death
preceded by multinucleation' or 'cell death occurring during
metaphase', which are more precise and more informative.
NCCD tentative definitions of Atypical
Cell Death Modalities: Examples
• 'Anoikis‘ (Gr. Homelessness)
Apoptosis induced by the loss of the attachment to the substrate or
to other cells.
Specific form of induction, but molecular mechanisms of anoikisassociated cell death match those activated during classical
apoptosis.
i
NCCD acknowledges the use of this term for historical reasons.
But do other modalities of cell death occur in vivo following
detachment? Are there forms of anoikis refractory to caspase
inhibitors and/or others that manifest necrotic features.
19
NCCD tentative definitions of Atypical
Cell Death Modalities: Examples
• 'Excitotoxicity'
•
A form of cell death occurring in neurons challenged with excitatory amino
acids, such as glutamate
•
Leads to the opening of the N-methyl-D-aspartate Ca2+-permeable
channel, followed by cytosolic Ca2+ overload and activation of lethal
signaling pathways
pathways.
•
Excitotoxicity seemingly overlaps with other types of death such as
apoptosis and necrosis (depending on the intensity of the initiating
stimulus).
•
Involves MMP as a critical event with presence of common regulators such
as nitric oxide.
•
Not be considered as a separate cell death modality.
NCCD tentative definitions of Atypical
Cell Death Modalities: Examples
• 'Entosis'
•
Originally described as a form of 'cellular cannibalism' in lymphoblasts from patients
with Huntington's disease.
•
Reported as a new cell death modality in which one cell engulfs one of its live
neighbors, which then dies within the phagosome.
•
The most efficient cells in performing entosis are MCF-7 breast cancer cells, which
lack both caspase-3 and beclin-1 and hence are (relatively) apoptosis- and
autophagy-incompetent.
•
Entosis may be a default pathway that is unmasked exclusively when other catabolic
reactions are suppressed.
•
Entosis is not inhibited by Bcl-2 or Z-VAD-fmk, and internalized cells appear virtually
normal.
•
Later internalized disappear, presumably through lysosomal degradation and in rare
cases internalized cells are able to divide within the engulfing cell or are released.
•
Does cell-in-cell morphology (entosis) truly represents a novel cell death modality.
NCCD tentative definitions of Atypical
Cell Death Modalities: Examples
• 'Pyroptosis'
•
Described in macrophages infected with Salmonella typhimurium.
•
Involves the apical activation of caspase-1 (but not of caspase-3), a
protease that is mostly known as interleukin-1 (IL-1 )-converting enzyme.
•
Caspase-1 activation induced by S.
S typhimurium (and by other pathogens
such as Pseudomonas aeruginosa and Shigella flexneri) occurs through
Ipaf and Apaf-1-related NLR protein.
•
Leads to the release of IL-1 (which is one of the major fever-inducing
cytokines or pyrogens) and of IL-18, it may play a relevant role in both local
and systemic inflammatory reactions.
•
Macrophages undergoing pyroptosis not only exhibit morphological features
that are typical of apoptosis, but also display some traits associated with
necrosis.
20
NCCD tentative definitions of Atypical
Cell Death Modalities: Examples
• 'Pyronecrosis'
Nalp3 and ASC are involved in the necrotic cell death of
macrophages infected by S. flexneri.
Associated with the release of HMGB-1, caspase-1 and IL-1 , which
py
is called pyronecrosis.
Pyronecrosis and pyroptosis are distinguished based on the fact that
the latter (but not the former) requires caspase-1.
Yet to be determined whether RIP1 is implicated in pyronecrosis, as
well as whether pyroptosis and pyronecrosis play any role outside of
the innate immune system.
Yet another novel cell death program:
Neutrophil extracellular traps (NETs)
Extracellular structures composed of intact chromatin decorated with
granule proteins that bind and kill microorganisms (Gram + and –
and fungi)
•
•
•
•
•
•
Nuclei of neutrophils lose their shape—euchromatin and
heterochromatin homogenize
Nuclear envelope and granule membranes disintegrate and mix
NETs released as cell membrane breaks
Death process distinct from apoptosis and necrosis: “Netosis?”
Depends on generation of ROS by NADPH oxidase
Neutrophils can be antimicrobial by ROS formation in:
– Intraphagosomal killing in live neutrophils
– NET mediated killing after their deaths.
Fuchs, T. A. et al. J. Cell Biol. 2007;176:231-241. Novel cell death program
leads to neutrophil extracellular traps.
Neutrophil extracellular traps (NETs)
Extracellular structures composed of chromatin and granule proteins that bind
and kill microorganisms.
Figure 1. Neutrophils die an active form of cell death to release NETs
Fuchs, T. A. et al. J. Cell Biol. 2007;176:231-241
Copyright ©2007 Rockefeller University Press
21
Questions for Lecture 2
About the Questions.
Cell Injury Questions from Lectures 1-4
come from two sources. Those
designated with this red # and ISU/Aub
at the bottom of the page were created
and contributed byy residents and facultyy
of Iowa State University and Auburn
University listed as a group. Æ
Answers for these appear at the end of
the question pages. Page numbers refer
to Pathologic Basis of Veterinary
Disease.
Other questions came from the AFIP web
site.
Questions from ISU/Aub
Contributed by:
Dr. Alicia Olivier
Dr. Katherine Gibson-Corley
Dr. Brandon Plattner
Dr. Darin Madson
Dr. Jodi Smith
Dr. Rachel Derscheid
Dr Charlie Johnson
Dr.
Dr. Molly Murphy
Dr. Tatjana Lazic
Dr. Ann Predgen
Dr. Aaron Lehmkuhl
Dr. Angela Pillatzki
Dr. Lisa Pohlman
Dr. Pete Christopherson
Dr. Brandon Brunson
Dr. Leah Ann Kuhnt
Dr. Kellye Sue Joiner
Dr. Beth Spangler
Dr. Elizabeth Whitley
49. The protein that can bind to and activate Apaf-1 to initiate apoptosis is :
A. Bcl-2
B. ICAD
C. Caspase-3
D Caspase
D.
Caspase-9
9
E. Cytochrome c
49. E Slausson & Cooper, pp. 41-42, 2002 2004 c
14. All are inhibitors of apoptosis (IAP) EXCEPT:
A. XIAP
B. NAIP
C. Apaf-1
D. Apollon
E. Survivin
14. C: Vet Pathol 41:599-604, 2004; Pathologic Basis of disease, 7th ed., 2005
22
10. Principal mechanisms by which cytotoxic T lymphocytes kill their targets are
perforin-granzyme-dependent killing and:
A. The Fenton reaction
B. Opsonization and phagocytosis
g
p
killing
g
C. Fas-Fas ligand-dependent
D. Reactive oxygen species-dependent killing
E. Complement activation and formation of “membrane attack complex”
10. C: Robbins and Cotran, Pathological Basis of Disease, p. 218, 2005. 2006
18. All are downstream targets of p53 in apoptosis EXCEPT:
A. Atm
B. Noxa
C. Apaf-1
D. Perp-1
E. PUMA
18. A: J Pathol 2005:205:206-220. 2006
22. All are true regarding Survivin EXCEPT:
A. Causes apoptosis
B. Expressed in adult thymus
C. Essential for proper cell division
D. Inhibitor of apoptosis (IAP) gene family
E. Expressed during embryonal development
22. A: Vet Path 41:599-607, 2004. 2006
23
36. In the intrinsic (mitochondrial) pathway of apoptosis, cytochrome c leaks
from the mitochondria, binds to Apaf-1 in the cytosol, and the resulting complex
activates:
A. Caspase-9
B Granzyme B
B.
C. Fas-associated death domain (FADD)
D. FLIP
36. A: Robbins and Cotran, Pathological Basis of Disease, pp. 29-30, 2005. 2006
13. All of the following are pro-apoptotic EXCEPT:
A. Bak
B Bid
B.
C. Bim
D. Bcl-x
E. C&D
13. D. Robbins and Cotran, p. 29-30 2007
14. Which of the following are important in recognition of apoptotic cells for
phagocytosis?
A. C3b
B. Thrombospondin
C. Thrombomodulin
D. Phosphatidylserine
E. B&D
14. E. Robbins and Cotran, p. 27 2007
24
25. What inhibits Fas-FasL mediated apoptosis by binding to pro-caspase 8?
A. bid
B. FLIP
C. bcl-x
D. NF-kB
E. TRADD
25. B. Robbins and Cotran, p. 29 2007
37. All of the following are true regarding apoptosis EXCEPT:
A.
Apaf-1 inactivates Caspase-9
B. Caspase-8 and Caspase-9 are initiator caspases
C. Caspase-3 and Caspase-6 are executioner caspases
D. Typically does not initiate an inflammatory response
E. B&C
37. A. Robbins and Cotran, p. 26, 30-31 2007
50. The pivotal event in the mitochondrial pathway of apoptosis is:
A. Transcription of Bax and Bak
B. Activation of APAF-1
C. Activated caspase-9 cleavage of caspase-3 and caspase-7
D. Mitochondrial outer membrane permeabilization
E. Sequestration of Smac and Omi in the mitochondrial
intermembrane space
50. D. Science 310:66-7, 2005 2007
25
15. Regarding apoptosis, which of the following statements are true?
1. Affected cells are smaller in size
2. Chromatin condensation is common
3. Apoptotic cells are usually phagocytized by neutrophils
4. It does not occur in pathologic conditions
5. The intrinsic pathway is initiated by activation of cell surface
death receptors
A. 1
B. 1, 2
C. 1, 2, 3
D. 1, 2, 3, 4
E. 1, 2, 3, 4, 5
15. B. Robbins and Cotran, p. 26-69 2008
27. All of the following are morphological features of apoptosis
EXCEPT:
A. Cell shrinkage and convolution
B. Intact cell membrane
C. Pyknosis
D. Karyorrhexis
E. Karyolysis
27. E. Toxicologic Pathology 35:495-516, 2007 2008
#1
Which features describe caspase enzymes of the apoptotic process?
1.
2.
3.
4.
5
5.
A.
B.
C.
D.
E.
Cysteine endopeptidase
Enzymatic activity at aspartic acid residues
Enzymatic activity between cysteine dimers
May undergo autocatalytic hydrolysis
Function as a transferase enzyme
1, 2
1, 2, 3
2, 3, 4
1, 2, 4
2, 3, 5
ISU/Aub
26
#10
A sequence of early events in acute hypoxic cell injury is:
1.
2.
3.
4.
5
5.
A.
B.
C.
D.
E.
Stimulation of phosphofructokinase
Cessation of aerobic oxidative phosphorylation
ATP levels decrease
Depletion of glycogen stores
Accumulation of intracellular lactate and inorganic phosphate
2, 1, 3, 5, 4
2, 3, 1, 4, 5
3, 2, 4, 1, 5
3, 4, 1, 2, 5
4, 3, 2, 1, 5
ISU/Aub
#18
During reperfusion injury, which processes DO or DOES NOT contribute to
further cell damage as a result of calcium (Ca++) ion influx into injured
cells (PBVD ed.4 p. 18)?
1.
2.
3
3.
4.
5.
A.
B.
C.
D.
E.
Direct cell membrane damage by the calcium ions
Lipid membrane damage due to activation of phospholipases
Generation of arachidonic acid
Cytoskeleton breakdown due to protease activation
Chromatin degradation due to endonuclease activation
1
1, 2
1, 2, 3
2, 3, 4, 5
All of these are mechanisms of calcium-mediated damage
ISU/Aub
#24
All of the following molecules are pro-apoptotic EXCEPT:
A. Bak
B. Bax
C Brm
C.
D. Bcl-x
E. Apaf-1
ISU/Aub
27
#39
The components of the apoptosome include all EXCEPT: (PBVD ed.4, p.
31)
A. Cytochrome C.
B. Pro-caspase 9.
C. Pro-caspase 8.
D. APAF-1.
ISU/Aub
#53.
The activation of cytoplasmic DNAase is controlled by which of the
following: (PBVD p. 32)
A. Caspase 9
B. Caspase 8
C. Caspase 6
D. Caspase 3
ISU/Aub
#64
Which caspases are considered the initiator caspases in both the extrinsic
and intrinsic apoptotic pathways? (PBVD ed.4 p. 31)
1.
2.
3.
4
4.
5.
A.
B.
C.
D.
E.
Caspase 8
Caspase 9
Caspase 10
Caspase 11
Caspase 12
1,2.
2,3.
1,4,5.
2,3,4,5.
1,2,3,4,5.
ISU/Aub
28
#68
Which of the following ultrastructural cell changes occur in apoptosis?
(PBVD ed.4, p. 17)
1.
2.
3.
4
4.
5.
A.
B.
C.
D.
E.
Formation of cytoplasmic blebs
Leakage of cellular components
Nuclear chromatin condensation
Nuclear fragmentation
Phagocytosis of cellular fragments
1,2.
1,2,3.
1,2,3,4.
1,2,3,4,5.
3,4,5*
ISU/Aub
#74
FLIP inhibits apoptosis by: (PBVD ed.4, p. 30)
A. Binding procaspase-8 and preventing its activation
B. Binding FasL and preventing it from binding FAS
C Preventing the cross
C.
cross-linking
linking of Fas
D. Binding procaspase-3 and preventing its activation
E. Sequestering cytochrome c
ISU/Aub
#76
In the intrinsic pathway of apoptosis what is the main protein responsible
for initiating apoptotic signaling pathways? (PBVD ed.4, p.30-31)
A. Fas
B. Bax-1
C. Bcl-2
D. Pro-caspase 9
E. Cytochrome c
ISU/Aub
29
#78
Regarding the extrinsic apoptosis pathway, which is/are NOT TRUE? (PBVD
ed.4, p. 30)
1.
2.
3.
4
4.
5.
A.
B.
C.
D.
E.
Mediated by Fas and FasL binding
Can be inhibited by FLIP
FADD activates procaspase 9
Some virus produce FLIP and inhibit extrinsic pathway
3 or more FAS are combined to make binding site called FADD
1
2
3
4
4 and 5
ISU/Aub
#89
Anti-apoptotic members of the Bcl—2 protein family include: (PBVD ed.4,
p. 30)
1.
2.
3.
4
4.
5.
A.
B.
C.
D.
E.
Bim
Bcl-2
Bcl-x
Bax
Bak
1,2
2,3
1,2,3
2,3,4
3,4,5
ISU/Aub
#93
All of the following are histopathologic characteristics of apoptotic necrosis
EXCEPT: (PBVD ed.4, p. 32)
A. Inflammation is present.
B. Cytoplasm is fragmented.
C. Chromatin is condensed.
D. Individual cells are shrunken.
E. Apoptotic bodies are present within adjacent cells and
phagocytes.
ISU/Aub
30
#104
Which of the following mechanisms promote apoptosis? (PBVD ed.4, pp. 2931)
1.
2.
3.
4
4.
5.
A.
B.
C.
D.
E.
Activation of the type I tumor necrosis factor receptor.
Activation of Bcl-2 proteins.
Activation of executioner caspases by cytotoxic T lymphocytes.
Binding of CD95 by Fas ligand
ligand.
Elaboration of cytochrome c by mitochondria.
1,2.
1,2,3.
3,4,5.
1,2,3,4.
1,3,4,5.
ISU/Aub
#110
In the intrinsic pathway of apoptosis, which enzyme is released following
increased mitochondrial permeability? (PBVD ed.4, p 31)
A. Apaf-1
B. Cytochrome-c
C. Bcl-2
D. Pro-caspase 9
E. A. and B.
ISU/Aub
#112
The following are executioner caspases EXCEPT: (PBVD ed.4, p. 30)
1.
2.
3.
4.
A.
B.
C.
D.
E.
Caspase 3
Caspase 6
Caspase 8
Caspase 9
1
2
1, 4
1, 2.
3, 4.
ISU/Aub
31
#129
Which are pro-apoptotic proteins? (PBVD ed.4, pp. 30-31)
1.
2.
3.
4.
5
5.
A.
B.
C.
D.
E.
Bcl-2.
Bcl-x.
Bak.
Bax.
Bim
Bim.
1,2.
1,2,3.
1,2,3,4.
1,2,3,4,5.
3,4,5*
ISU/Aub
Answers to ISU/Aub questions for lecture 2.
•
•
•
•
•
•
•
•
•
•
•
•
•
1. D
10. B
18. A
24. D
39. C
53. D
64. A
68. E
74. A
76. E
78. C
89. B
93. A
•
•
•
•
104.
110.
112.
129.
E
B
E
E
ISU/Aub
32