02_CELL INJURY by prof ook ibrahim II-1.pptx

Cell injury

1. CELL INJURY II Types of cell injury: Reversible vs irreversible Cell death Mechanisms of cell death: Necrosis; Apoptosis; Necroptosis; Pyroptosis ; Ferroptosis

2. Reversible injury This is characterized by functional and structural alterations which are correctable if the damaging stimulus is removed. It occurs usually in early stages of exposure to injurious stimulus or mild forms of injury. The features include: Generalized swelling of the cell and its organelles due to influx of water, blebbing of plasma membrane, detachment of ribosomes from ER, clumping of nuclear chromatin. All these result from ATP depletion resulting from hypoxia or direct damage to mitochondria. Fatty change: Occurs in organs that are involved in lipid metabolism.

3. Reversible injury Morphological changes Gross: Cellular swelling resulting in organ pallor, increased weight, increased turgor . Light microscopic: Hydropic change or vacuolar degeneration Electron microscopic: Plasma membrane alterations: blebbing, blunting, loss of microvilli Mitochondria swelling and appearance of small amorphous densities. Accumulation of myelin figures Dilation of ER with detachment of polysomes, etc

4. Irreversible cell injury (Cell death) There are two main types (mechanisms) of cell death: Necrosis Apoptosis They have different mechanisms, different morphology, and have different roles in physiology and pathology. Necrosis is usually associated with severe damage to the mitochondria with ATP depletion and rupture of plasma and lysosomal membranes. Apoptosis is a regulated cell death mediated by defined molecular pathways that are activated under specific circumstances and kill the cell. Also referred to as programmed cell death. Other mechanisms include necroptosis (programmed necrosis), pyroptosis , ferroptosis

5. Necrosis It is a pathologic process that results from severe injury. It is characterized by denaturation of cellular proteins, leakage of cellular contents through damaged membranes, inflammation, and enzymatic digestion of lethally injured cell. Main causes include hypoxic-ischemic injury, microbial toxins, burns, physical and chemical injury.

6. Necrosis General morphology: Nuclear changes – due to breakdown of DNA Karyolysis – Fading of the basophilia of chromatin. Pyknosis – Nuclear shrinkage and increased basophilia. Karyorrhesis – when pyknotic nucleus undergoes fragmentation, and the nucleus later disappears. Cytoplasmic changes: Increased eosinophilia – this is due loss of cytoplasmic RNA and also accumulation of denatured cytoplasmic proteins.

7. Necrosis Morphological patterns of tissue necrosis Coagulative necrosis: Architecture of the tissue is preserved for few days. The affected tissue is firm in consistency. There is denaturation of structural proteins as well as enzymatic proteins preventing proteolysis of the dead cells. Necrotic cells are ultimately broken down by lysosomal enzymes from invading leukocytes. Ischemia induces coagulative necrosis in most organs expect in the brain. A localized area of ischemic necrosis is termed infarct.

8. Morphological patterns of tissue necrosis Liquefactive necrosis: It is characterized by enzymatic digestion of the dead cells. Seen in focal bacterial and sometimes fungal infections. Microbes stimulate accumulation of leukocytes. Lysosomal enzymes from the infiltrating leukocytes digest the dead cells. The necrotic material is usually creamy yellow (pus). Hypoxic-ischemic deaths in the CNS often manifest as liquefactive necrosis (possibly due to abundant microglia).

9. Morphological patterns of tissue necrosis Gangrenous necrosis A form of coagulative necrosis. Usually applied to a limb, especially lower limb, that has lost its blood supply. It becomes liquefactive necrosis with superimposed bacterial infection (due enzymes in bacteria and invading leukocytes)…referred to as wet gangrene. Caseous necrosis A form of coagulative necrosis. Most often seen in tuberculous infection. The caseous (cheese-like) material has a friable yellowish white appearance grossly. It has structureless amorphous eosinophilic appearance on light microscopy.

10. Morphological patterns of tissue necrosis Fat necrosis This is a focal area of fat destruction. Usually due to release of activated pancreatic lipases into the pancreas and peritoneal cavity. It occurs in acute pancreatitis. It can also occur in trauma or other injuries, e.g., breast. Fibrinoid necrosis This is a special form of vascular damage, immune complex vasculitis

11. Necrosis Outcome of necrosis The necrotic cells are degraded by lysosomal enzymes and the debris are phagocytosed by leukocytes. Alternatively, there is deposition of calcium salts and other minerals on the necrotic cells and cellular debris if they are not reabsorbed. This is termed dystrophic calcification.

12. Apoptosis It is a form of cell death that is induced by a tightly regulated suicide program in which cells destined to die activate intrinsic enzymes that degrade the cell’s genomic DNA and nuclear and cytoplasmic proteins. It is a unique mechanism of cell death that is distinct from necrosis. It is also termed programmed cell death especially during embryogenesis.

13. Causes of apoptosis It occurs as part of normal physiologic processes and as a pathophysiologic mechanism of cell loss in many diseases (pathologic) In physiological processes: It serves to eliminate cells that are no longer needed or mechanism to maintain a constant number of various cell populations in tissues. E.g.; Removal of excess cells during development – required for involution of primordial structures. Involution of hormone-dependent tissues on hormone withdrawal; e.g.; during menstrual cycle, regression of lactating breast after weaning. Cell turnover in proliferating cell populations Elimination of potentially harmful self-reactive lymphocytes Death of host cells that have served their purpose.

14. Apoptosis in pathologic context It serves to eliminate cells that injured beyond repair without eliciting inflammatory response (which can cause tissue damage). The pathologic states include: DNA damage – through radiation injuries, anticancer drugs, free radical injuries, etc. It is protective because of the consequences of the DNA damage including cancer-induced mutations. During certain infections, especially viral infections, to eliminate viral infected cells. Killing of tumor cells. Pathologic atrophy in parenchymal organs after duct obstruction. E.g.; parotid gland.

15. Morphologic and biochemical changes in apoptosis Cell shrinkage Chromatic condensation: most characteristic feature of apoptosis. Formation of cytoplasmic blebs and apoptotic bodies. Phagocytosis of apoptotic bodies by macrophages. Note that it is not usually apparent on histologic sections because formation of apoptotic bodies are rapid and they are quickly cleared by macrophages. Also there is inflammatory response.

16. Mechanisms of apoptosis Apoptosis is mediated by caspases. Caspases are named because contain cysteine in their active site and cleave proteins after aspartatic residues. Caspases occur in inactive form that must cleaved and activated, and in turn activate further caspases until the terminal caspases trigger cellular fragmentation. Process of apoptosis can be divided into initiation phase and execution phase. Regulation of these caspases depends on the fine balance between abundance and activity of pro-apoptotic and anti-apoptotic proteins. Two distinct pathways converge on caspase activation: The mitochondrial pathway and the death receptor pathway.

17. Mechanisms of apoptosis The mitochondrial pathway (intrinsic) This is responsible for most physiologic and apoptosis in pathologic conditions. It results from increased permeability of mitochondrial outer membrane – leading to release of pro-apoptotic molecules (e.g., cytochrome c) from mitochondrial intermembrane into the cytoplasm. Release of cytochrome c is determined by integrity of outer mitochondrial membrane. Integrity of outer mitochondrial membrane is tightly controlled by BCL2 family of proteins (named after BCL2 gene – overexpressed gene in B cell lymphoma).

18. Mechanisms of apoptosis The mitochondrial pathway (intrinsic) contd Members of BCL family of protein are divided into 3 based on their pro-apoptotic and anti-apoptotic function and BCL2 homology (BH) domains they possess: Anti-apoptotic: BCL2, BCL-XL, and MCL1. They possess four BH domains (BH1-4). These proteins reside at outer mitochondrial membrane and keeping it impermeable and prevent leakage of cytochrome c into the cytosol. Pro-apoptotic: BAX and BAK. They possess the first three BH domains (BH1-3). These proteins oligomerize on activation within outer mitochondrial membrane and enhance its permeability. Regulated apoptosis initiators: BAD, BIM, BID, Puma, and Noxa . They contain the third of the four BH domains (BH3-only). Their activity is modulated by sensors of cellular stress and damage. They can initiate apoptosis when upregulated and activated.

19. Mechanisms of apoptosis The mitochondrial pathway (intrinsic) contd Growth factors and survival signals stimulate production of anti-apoptotic proteins such as BCL2 – which prevent leakage of cytochrome c by strengthening outer mitochondrial membrane. Lack of survival signals or DNA damage will lead to upregulation of BH3-only proteins, which in turn directly activate pro-apoptotic family BAX and BAK that form oligomers that insert into the inner mitochondrial membrane and allow cytochrome c to leak into cytosol. Also BH3-only proteins also block the function of anti-apoptotic proteins, BCL2 and BCL-XL.

20. Mechanisms of apoptosis The mitochondrial pathway (intrinsic) contd The released cytochrome c binds to APAF-1 (apoptosis-activating factor-1) to form apoptosome . The apoptosome binds to and activate caspase-9, an initiator caspase of mitochondrial pathway. Active caspase-9 triggers a cascade of activation of other procaspases such as caspase 3 which mediate the execution phase.

21. Mechanisms of apoptosis The death receptor-initiated pathway (Extrinsic) Initiated by engagement of plasma membrane death receptors. Death receptors are members of tumor necrotic factor receptor family. They have a cytoplasmic death domain that is essentially for delivering apoptotic signals. Typical examples are type 1 TNF receptor (TNFR1 ) and a related protein called Fas (CD95). Binding of Fas ligand ( FasL ) to Fas will lead to dimerization of the cytoplasmic death domains that will bind adapter protein called FADD ( Fas –associated death domain. This will bind inactive caspase-8 or -10. Active caspase-8 will trigger further activation of downstream caspases up to caspase 3, which is an execution caspase.

22. Mechanisms of apoptosis Execution phase Initiator caspase for intrinsic is caspase-9 while those of extrinsic are caspases-8 and 10. These initiator caspases trigger activation of execution caspases such as caspase 3, caspase-6. Caspase-3 cleaves an inhibitor of a DNase, making DNase active and result in DNA degradation.

23. Mechanisms of apoptosis Removal of dead cells Apoptotic cells and their fragments are phagocytosed by macrophages. Apoptotic cells express phosphotidylserine on their membranes that are recognized by macrophages. Apoptotic cells may also be coated with natural antibodies and proteins of the complement system, especially C1q , that are recognized by macrophages. Process of apoptotic cell phagocytosis is called efferocytosis. Note that production of pro-inflammatory cytokines is reduced in macrophages that have engulfed apoptotic cells. This will limit inflammatory reaction in apoptosis.

24. Other forms of cell death Necroptosis: Hybrid with features of necrosis and apoptosis. It resembles necrosis by morphology but it is triggered by signal transduction pathway, biding of ligand to TNFR1. Also called programmed necrosis, and caspase-independent programmed cell death. Pyroptosis : A form of apoptosis accompanied by release of fever-inducing cytokine, IL-1. Ferroptosis: A distinct form of cell death that is triggered by excessive intracellular level of iron or reactive oxygen species. This results in lipid peroxidation.