4.pathology apoptosis2016

Pathology ---Cell Injury, Death And

Adaptation

Xi’an Jiaotong University

2017.1.2017.1.

XI’AN JIAOTONG UNIVERSITY

Programmed Cell Death (PCD) Refers to processes that are lethal to individual cells

and are regulated by pre-existing signaling pathways.

PCD is part of the balance between the life and

death of cells and determines that a cell dies when it is

no longer useful or when its survival may be harmful to

the larger organism.


Apoptosis A pathway of cell death that is induced by a tightly

regulated suicide program in which cells destined to die, activate intrinsic enzymes that degrade the cells’own nuclear DNA and nuclear and cytoplasmic proteins Apoptosis is a form of PCD that relies exclusively on the caspase cascade Originally, PCD was synonymous with apoptosis. However, mutant

mice lacking the key elements of the apoptotic machinery develop almost

normally. This observation indicated that alternatives to apoptosis exist.

Thus, a number of mechanisms of PCD have been identified


Morphologic changes

Cell shrinkage

Recall that in other forms of cell injury, an early feature is cell swelling,

not shrinkage

Chromatin condensation and fragmentation.

The most characteristic feature of apoptosis

Formation of cytoplasmic blebs and apoptotic bodies.


Phagocytosis of apoptotic cells or cell bodies, usually by macrophages.

Histologically, apoptotic cell appears as a round or oval mass of intensely eosiniphilic cytoplasm (H&E staining) with or without fragments of dense nuclear chromatin. Because these pieces are quickly formed and phagocytosed, without eliciting inflammation, even substantial apoptosis may be more difficult to detect histologically.


Sequence of ultrastructural changes in apoptosis of glandular epithelial cells. The earliest change is segregation of the chromatin in sharply circumscribed masses that abut nuclear envelop (1). Convolution of the nuclear and cell outlines precedes fragmentation of the nucleus and budding of the cell as a whole to produce membrane-bound apoptotic bodies of varying size and structure(2), which are phagocytosed by adjacent epithelial cells(3) and macrophages(4) and degraded within lysosomes(5). Finally, the bodies are reduced to unrecognizable residues(6). An occasional apoptotic body escapes phagocytosis and is shed into the gland lumen(7) where it undergoes degenerative changes similar to those occurring in necrosis(8)


Early apoptosis of acinar cell in rat pancreas after duct ligation. Note granular fibrillar core remnant of nucleolus adjacent condensed chromatin(arrow) adjacent cells appear normal(x6200)

Apoptotic HeLa cell after addition of actinomycin D to cultured medium(SEM x9000)


Early apoptotosis of rat prostatic epithelial cell after castration.Note irregular cell outline, basal position of cell in epithelium, segregated chromatin in nuclear fragments and whorling of endoplasmic reticulum(x8200)

Intact extracellular condensed apoptotic bodies, some containing characteristic nuclear fragments, derived from transplanted EMT6 sarcoma cell after mild hyperthermic treatment(x10,000)


Crowded condensed intraepithelial apoptotic bodies derived from pancreas acinar cell after subcutaneous cerulein injection(x8,300).

Well preserved large apoptotic body with characteristic nuclear fragment phagocytosed by a tubular epithelial cell in rat kidney after ureteric ligation(x4500)


Partly degraded apoptotic bodies, one with nuclear fragments(arrow), in intraepithelial macrophage in rat pancreas after duct ligation(x7500)

Well-preserved hepatocyte-derived apoptotic body with condensed cytoplasm, crowded organelles, and characreistic nuclear fragment phagocytosed by Kupffer cell in rat liver after portal vein branch ligation


Multiple partly degraded apoptotic bodies in intraepithelial macrophage in rat pancreas after duct ligation(x6700)

Residual bodies in cytoplasm of intraepithelial macrophage in rat pancreas after duct ligation(x7500)


Luminal apoptotic bodies in lactating mouse breast after weaning(A),intact apoptotic bodies with characteristic nuclear fragment. (B), Partly degraded apoptotic body with nuclear fragment showing decreased density and loss of membrane integrity.(C) , Degraded apoptotic body showing membrane loss and dispersal of nuclear fragments(arrows) and organelles.(x3000, 3900, 5200)


Apoptotic cells and bodies in rat liver after portal vein branch ligation. Note apoptotic hepatocyte with characteristic crescentic chromatin clumps in nucleus(1), budding cells(2), and typic apoptotic body with basophilic chromatin dot(3). Also note numerous small inconspicuous phagocytosed apoptotic bodies without nuclear fragments in the backgroud

Clusters of apoptotic bodies in rat parotid after duct ligation. Some small clusters may represent bodies derived from a single cell(short arrow). Larger clusters are likely to be phagocytosed apoptotic bodies within intraepithelial macrophages(long arrow)


Aoptosis of epithelial cells(arrow) in ductules of normal human breast

Apoptotic bodies in intraepithelial macrophages in late secretory human endometrium


Cause of apoptosis

Apoptosis in physiologic situations Death by apoptosis is a normal phenomenon that serves

to eliminate cells that are no longer needed, and to maintain

a steady number of various cell populations in tissue

The destruction of cells during embryogenesis Fetal development involves the sequential appearance and regression of many evolutionary relics.


Involution of hormone-dependent tissue upon hormone withdrawal menstrual cycle, lactating breast after weaning

Cell loss in proliferating cell populations turnover of intestinal crypt epithelia, bone marrow lymphocytes,

Elimination of potentially harmful self-reactive lymphocytes either before or after they have completed their maturation

Death of host cells that have served their useful purpose neutrophils, lymphocytes at the end of response

Cell death induced by cytotoxic T lymphocytes.


Apoptosis in pathologic situations

Apoptosis eliminates cells that are injured beyond repair without eliciting a host reaction, thus limiting collateral tissue damage.

DNA damage radiation, cytotoxic anticancer drugs, hypoxia, either

directly or via free radicals

Accumulation of misfolded proteins excessive amount of these proteins in ER leads to a condition called ER

stress, which culminates in apoptotic cell death, which may cause

several degenerative disease of CNS


Cell death in certain infections Directly such as adenovirus –cytolysis,indirectly cell mediated

immune response ； Conversly, HPV infection

Pathologic atrophy in parenchymal organs after duct obstruction, such as in pancreas, paroit gland, and kidney


Mechanism of Apoptosis

Apoptosis results from the activation of enzymes called

caspase.

Two distinct pathways converge on caspase activation:

the mitochondrial pathway and the death receptor pathway


The two pathways of apoptosis differ in their induction and regulation, and both culminate in the activation of "executioner" caspases. The induction of apoptosis is dependent on a balance between pro- and anti-apoptotic signals and intracellular proteins. The figure shows the pathways that induce apoptotic cell death, and the anti-apoptotic proteins that inhibit mitochondrial leakiness and cytochrome c-dependent caspase activation and thus function as regulators of mitochondrial apoptosis.


NecroptosisA hybrid form of cell death that shares aspects of both necrosis and apoptosis （ programmed necrosis)

Morphologically, and to some extent biochemically, it

resembles necrosis, both characterized by loss of ATP,

swelling of the cell and organelles, generation of ROS,

release of lysosomal enzymes and ultimately rupture of

the plasma membrane as discussed early


Mechanistically, it is triggered by genetically programmed

signal transduction events that culminate in cell death. In this

respect it resembles programmed cell death---the hallmark of

apoptosis. In sharp contrast to apoptosis, the genetic

program that drives necroptosis does not result in caspase

activation (caspase independent programmed cell death)


TNF-induced formation of apoptotic and necroptotic signaling complexes. nature immunology 2011,12(12):1143


Necroptosis is induced by various stimuli. Different necroptotic stimuli are recognized or sensed by specific receptors or sensors on the cell surface or inside cells


Pyroptosis Bearing some biochemical similarities with apoptosis and releasing fever producing cytokine IL-1 Initially described in immune cells during antimicrobial response, is a caspase-1 or caspase-11-dependent regulated type of cell death that plays a central role in inflammation and immunity


Anoikis Activated by Loss of Cell Attachments Anoikis (Greek: “homelessness”) is a variety of apoptosis

that occurs in epithelial cells and is caused by loss of cell

adhesion or inappropriate cell adhesion Correct binding of

a cell to the ECM helps to determine whether that cell is in

its appointed location


Mechanisms of anoikis. A. Normal. Under normal circumstances, epithelial cells are bound to their native ECM by transmembrane molecules, including α- and β-integrins. These molecules activate survival signals and block both intrinsic and extrinsic apoptotic signaling pathways. B. Loss of attachment. When the cell’s integrins are not bound, or not bound by the appropriate ECM moieties, their survival signals are eliminated. Then, activation of apoptosis by death receptor signaling is no longer blocked, and apoptosis may proceed.


Entosis Is a Cell-Eat-Cell Form of Cell Death Entosis is a type of cellular cannibalism in which cells

that are not professional phagocytes engulf nearby living

cells. Aggressor cells may engulf cells of either the same or

other lineages. For example, hepatocytes may ingest and

destroy autoreactive T lymphocytes, thus inhibiting

experimental autoimmune liver disease. More often, Entosis

is seen in tumors


SUMMARY:

Morphologic Alterations in Injured Cells

Reversible cell injury:

Cell swelling, fatty change, plasma membrane blebbing and

loss of microvilli, mitochondrial swelling, dilation of ER,

eosinophilia (due to decreased cytoplasmic RNA).


Irreversible cell injury Necrosis: increased eosinophilia; nuclear shrinkage,

fragmentation, and dissolution; breakdown of plasma

membrane and organellar membranes; leakage and

enzymatic digestion of cellular contents(tissue pattern/

inflammation).

Apoptosis: nuclear chromatin condensation; formation of

apoptotic bodies (fragments of nuclei and cytoplasm)


Key concepts on apoptosis

● Regulated mechanism of cell death that serves to eliminate unwanted and irreparably damaged cells, with the least possible host reaction

● Characterized by enzymatic degradation of proteins and DNA, initiated by caspases; and by recognition and removal of dead cells by phagocytes


● Initiated by two major pathways:

Mitochondrial (intrinsic) pathway is triggered by loss of survival signals, DNA damage and accumulation of misfolded proteins (ER stress); associated with leakage of pro-apoptotic proteins from mitochondrial membrane into the cytoplasm, where they trigger caspase activation; inhibited by anti-apoptotic members of the Bcl family, which are induced by survival signals including growth factors. Death receptor (extrinsic) pathway is responsible for elimination of self-reactive lymphocytes and damage by cytotoxic T lymphocytes; is initiated by engagement of death receptors (members of the TNF receptor family) by ligands on adjacent cells.


Key concepts on necroptosis and pyroptosis

● Necroptosis resembles necrosis morphologically and apoptosis mechanistically as a form of programmed cell death● Necroptosis is triggered by ligation of TNFR1, and viral proteins of RNA and DNA viruses● Necroptosis is caspase-independent but dependent on signalling by the RIP1 and RIP3 complex (receptor-interacting

protein 1/3, RIP1/3)


● RIP1-RIP3 signally reduces mitochondria ATP generation, cause production of ROS, and permeabilize lysosomal membranes, thereby causing cellular swelling and membrane damage as occurs in necrosis.● Release of cellular contents evokes an inflammatory reaction as in necrosis● Pyroptosis occurs in cells infected by microbes. It involves activation of caspase-1 which cleaves the precursor form of IL-1 to generate biologically active IL-1. Caspase-1 along with closely related caspase-11 also cause death of the infected cell.


Autophagy A process in which a cell eat its own content, based on both the cargoes involved and how they arrive at Lysosomes, Autophagic degradation is generally divided into three categories ● Chaperone-mediated autophage (direct translocation across the lysosomal membrane by chaperone proteins) ● Microautophagy (inward invagination of lysosomal membrane for delivery)● Macroautophagy ( the major form of autophagy involving the sequestration and transportation of protein of cytosol in a double membrane bound autophagic vacuole


Types of autophagy. A. Macroautophagy. Cytoplasmic organelles are partially sequestered by an open membrane, the phagophore. Upon closure by fusion, the phagophore becomes an autophagosome, which then delivers its contents to a lysosome. Lysosomal enzymes degrade the contents to small molecules for reutilization. B. Microautophagy. Cytosolic cargoes are engulfed by invagination of the lysosomal membrane.The contents are then degraded by lysosomal enzymes. C. Chaperone-mediated autophagy. Proteins conjugated to chaperones (e.g., Hsc70) are recognized by lysosomal receptor proteins (LAMP-2A) and translocated to the lysosomal interior, where they are received by a second chaperone and then degraded. The original, extralysosomal chaperone survives to work further.


Autophagy. Cellular stresses, such as nutrient deprivation, activate autophagy genes (Atg genes), which initiate the formation of membrane-bound vesicles in which cellular organelles are sequestered. These vesicles fuse with lysosomes, in which the organelles are digested, and the products are used to provide nutrients for the cell. The same process can trigger apoptosis, by mechanisms that are not well defined.


CellularInjury

Microbiologic

Immunologic

GeneticPhysical

Chemical

Hypoxia NutritionAging

Causes of Cell Injury Causes of Cell Injury


Oxygen deprivation ----Hypoxia

Cause of Hypoxia:Reduced blood flow—ischemiaInadequate oxygenation—cardiorespiratory failureDecreased oxygen-carrying capacity of the blood---anemia or CO poisoning or severe blood loss

Hypoxia → ATP synthesis↓ (affects oxidative phosphorylation and ATP synthesis) → membrane damage → cell injury.


Physical agents Mechanical trauma; Extremes of temperatures

Abrasion(scratch) represents the tearing away of epidermal cells by fraction or crushing, such a defect may or may not penetrate the corium

Multiple Laceration of liver caused by laterial compression of thorax. There were no external wounds of fracture. Death resulted from hemoperitoneum contusion

Solar Dermatitis


Mechanisms by which ionizing radiation at low and high doses causes cell death

Radiation


p53-mediated apoptosis. p53 is recruited to areas of DNAdamage following external irradiation. Activated p53 activates p21WAF/CIP1, which halts cell cycle progression at the G1/S and G2/M transition points. If DNA damage is irreparable, p53 activates transcription of NOXA and PUMA, which alter the balance of proapoptotic and antiapoptotic Bcl-2 family proteins at the mitochondrial membrane in favor of apoptosis. The MPTP is opened leading to apoptosis.


Chemical agents Any chemical substance can cause injury, even normal

substances such as glucose or salt. Poisons can cause severe damage at the cellular level by

altering membrane permeability, osmotic homeostasis, or the integrity of an enzyme and result in the death of the whole organism.

eg.: air pollutants, insecticides, carbon monoxide, strong acids or alkali, and social “stimuli” such as alcohol, and other poisons such as arsenic, cyanide, melamine, even therapeutic drugs.


Infectious agentsVirus, Rickettsiae, Bacteria, Fungi, Parasites.

Immunologic reactions

May be lifesaving or lethal.Immune system defends against biologic agents; may lead to cell injury. e.g., hypersensitivity: anaphylaxis (asthma), autoimmune diseases (systemic lupus erythematosus, SLE), etc.


Nutritional imbalances Protein-calorie insufficiency; Specific vitamin deficiency; The excesses of nutrition: e.g. obesity markedly increases the risk for type 2

diabetes, AS and many disorders.


Genetic abnormalities

Chromosome abnormality -Down syndromeSingle base pair mutation– sickle cell anemia Mutations may cause alteration of functional proteins, influencing metabolism

Somatic mutations involved in activation of proto-oncogene and deletion of tumor suppressor gene may lead to cellular malignant transformation.


Mechanisms of cell injury

● The cellular response to injurious stimuli depends on the type of injury, its duration, and its severity.

● The consequences of cell injury depend on the type, status and adaptability of the injured cell.

● Cell injury results from different biochemical mechanisms acting on several essential cellular components


The principal biochemical mechanisms and sites of damage in cell injury. (1) ATP generation, largely via mitochondrial aerobic respiration; (2) Cell membrane integrity, critical to cellular ionic and osmotic homeostasis ;(3) Protein synthesis (4) The integrity of the genetic apparatus


Mitochondria damage Mitochondria are critical players in cell injury and cell death by all pathways.

Three major consequences of mitochondria damage

● Mitochondria damage often results in the formation of a high-

conductance channel in the mitochondria membrane, called mitochondrial

permeability transition pore

● Abnormal oxidative phosphorylation also leads to the formation of

reactive oxygen species,

● The mitochondria sequester between their outer and inner membranes

several proteins that are capable of activating apoptotic pathway.


Role of mitochondria in cell injury and death. Mitochondria are affected by a variety of injurious stimuli and their abnormalities lead to necrosis or apoptosis.


Influx of Sources and consequences of increased cytosolic calcium in cell injury.

● The accumulation of Ca2+ in mitochondria results in opening of the mitochondria permeability transition pore ●increased cytosolic Ca2+ activates a number of enzymes with potentially deleterious effects on cells ●increased intracellular Ca2+ levels also result in the induction of apoptosis by direct activation of caspases and by increasing mitochondrial permeability.


The generation, removal, and role of reactive oxygen species (ROS) in cell injury. The production of ROS is increased by many injurious stimuli. These free radicals are removed by spontaneous decay and by specialized enzymatic systems. Excessive production or inadequate removal leads to accumulation of free radicals in cells, which may damage lipids (by peroxidation), proteins, and deoxyribonucleic acid (DNA), resulting in cell injury.


Defects in membrane permeability

Mechanisms of membrane damage in cell injury. Decreased O2 and increased cytosolic Ca2+ typically are seen in ischemia but may accompany other forms of cell injury. Reactive oxygen species, which often are produced on reperfusion of ischemic tissues, also cause membrane damage


Damage to DNA and proteins Cells have mechanisms that repair damage to DNA, but if DNA damage is too severe to be corrected(e.g., after exposure to DNA damaging drugs, radiation, or oxidative stress), the cell initiates a suicide program that results in death by apoptosis


Aging Biological aging can be defined as a constellation of deleterious functional and structural changes that are inevitable consequences of longevity.

Importantly, aging must be distinguished from disease. Although the aging process may increase vulnerability to many diseases, it is independent of the pathogenesis of any specific illness. Biological longevity is generally interpreted as the extension of life well beyond the period of reproduction and childrearing


Life span of animals in their natural environment compared with that in a protected habitat. Note that both curves reach the same maximal life span.

Decrease in human physiologic capacities as a function of age.


Cellular aging the result of a progressive decline in the life span and functional capacity of cells.

The Hallmarks of Aging: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication


1960 Leonard Hayflik’s work by counting the number of times that population of cells had doubled . When the cells enter into senescence , they could remain viable but nonproliferating for as long as a year


Barbara McClintock 1983 Nobel Prize

Telomeres detected by FISH(left),( right) karyotype seen in left compared with that of cells have been deprived of TRF2


Role of large T antigen in circumventing senescence

Inactivation of both pRB and p53 is needed to ensure that these cells do not senesce in culture. This can be archived through the expression of the SV40 large T antigen in the target cells


Hutchinson-Guilford progeria. A 10-year-old girl shows the typical features of premature aging associated with progeria.


Interventions that Might Extend Human Health spanThe nine hallmarks of aging are shown together with those therapeutic strategies for which there is proof of principle in mice

4.pathology apoptosis2016

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