Chapter 2: Cell Injury, Cell Death, and Adaptations

Causes of Cell Injury

The Progression of Cell Injury and Death

It is useful to describe the basic alterations that occur in damaged cells before discussing the mechanisms that bring about these changes. All stresses and noxious influences exert their effects first at the molecular or biochemical level. There is a time lag between the stress and the morphologic changes of cell injury or death; understandably, the early changes are subtle and are only detected with highly sensitive methods of examination (Fig. 2.3). With histochemical, ultrastructural, or biochemical techniques, changes may be seen in minutes to hours after injury, whereas changes visible by light microscopy or the naked eye may take considerably longer (hours to days) to appear. As would be expected, the morphologic manifestations of necrosis take more time to develop than those of reversible damage. For example, in ischemia of the myocardium, cell swelling is a reversible morphologic change that may occur in a matter of minutes, and is an indicator of ongoing cellular damage that may progress to irreversibility within 1 or 2 hours. Unmistakable light microscopic evidence of cell death, however, may not be seen until 4 to 12 hours after onset of ischemia.

The sequential structural changes in cell injury progressing to cell death are illustrated in Fig. 2.4 and described later. Within limits, the cell can repair the alterations seen in reversible injury and if the injurious stimulus abates, may return to normalcy. Persistent or excessive injury, however, causes cells to pass the rather nebulous “point of no return” into irreversible injury and cell death. Different injurious stimuli induce death mainly by necrosis and/or apoptosis (see Fig. 2.4 and Table 2.1).

Necrosis

Necrosis is characterized by denaturation of cellular proteins, leakage of cellular contents through damaged membranes, local inflammation, and enzymatic digestion of the lethally injured cell. When damage to membranes is severe, lysosomal enzymes enter the cytoplasm and digest the cell. Cellular contents also leak through the damaged plasma membrane into the extracellular space, where they elicit a host reaction (inflammation). Some specific substances released from injured cells have been called damage-associated molecular patterns (DAMPs). These include ATP (released from damaged mitochondria), uric acid (a breakdown product of DNA), and numerous other molecules that are normally confined within healthy cells and whose release is an indicator of severe cell injury. These molecules are recognized by receptors present in macrophages and most other cell types, and trigger phagocytosis of the debris as well as the production of cytokines that induce inflammation (Chapter 3). Inflammatory cells produce more proteolytic enzymes, and the combination of phagocytosis and enzymatic digestion usually leads to clearance of the necrotic cells.

Necrosis-associated leakage of intracellular proteins through damaged plasma membranes and ultimately into the circulation is the basis for blood tests that detect tissue-specific cellular injury. Cardiac muscle cells, for example, express cardiac-specific variants of the contractile protein troponin, while bile duct epithelium expresses a specific isoform of the enzyme alkaline phosphatase and hepatocytes express transaminases. Necrosis of these cell types and associated loss of membrane integrity is reflected in increased serum levels of these proteins, which serve as biomarkers that are used clinically to assess and quantify tissue damage. Cardiac-specific troponins can be detected in the blood as early as 2 hours after myocardial cell necrosis, well before histologic evidence of myocardial infarction becomes apparent. Because of their sensitivity and specificity, serial measurement of serum cardiac troponins has a central role in the diagnosis and management of patients with myocardial infarction (Chapter 12).

It is useful to consider the possible events that determine when reversible injury becomes irreversible and progresses to necrosis. The clinical relevance of this question is obvious—if we can answer it, we may be able to devise strategies for preventing cell injury from having permanent deleterious consequences. Although the “point of no return,” at which the damage becomes irreversible, is still largely undefined, two phenomena consistently characterize irreversibility—the inability to reverse mitochondrial dysfunction (lack of oxidative phosphorylation and ATP generation) even after resolution of the original injury, and profound disturbances in membrane function. As mentioned earlier, injury to lysosomal membranes results in the enzymatic dissolution of the injured cell that is characteristic of necrosis.

Patterns of Tissue Necrosis

The discussion of necrosis has focused so far on changes in individual cells. When large numbers of cells die, the tissue or organ is said to be necrotic; thus, a myocardial infarct is necrosis of a portion of the heart caused by death of many myocardial cells. Necrosis of tissues has several morphologically distinct patterns, which are important to recognize because they provide clues about the underlying cause. Although the terms that describe these patterns are somewhat dated, they are often used and their implications are understood by pathologists and clinicians.

Morphology

Coagulative necrosis is a form of necrosis in which the architecture of dead tissue is preserved for a span of at least some days (Fig. 2.7). The affected tissue has a firm texture. Presumably, the injury denatures not only structural proteins but also enzymes and so blocks the proteolysis of the dead cells; as a result, intensely eosinophilic cells with indistinct or reddish nuclei may persist for days or weeks. Ultimately, the necrotic cells are broken down by the action of lysosomal enzymes derived from infiltrating leukocytes, which also remove the debris of the dead cells by phagocytosis. Ischemia caused by obstruction in a vessel may lead to coagulative necrosis of the supplied tissue in all organs except the brain (see next paragraph for explanation). A localized area of coagulative necrosis is called an infarct.

Liquefactive necrosis, in contrast to coagulative necrosis, is characterized by digestion of the dead cells, resulting in transformation of the tissue into a viscous liquid. It is seen in focal bacterial or, occasionally, fungal infections, because microbes stimulate the accumulation of leukocytes and the liberation of enzymes from these cells. The necrotic material is frequently creamy yellow because of the presence of leukocytes and is called pus. For unknown reasons, hypoxic death of cells within the central nervous system often manifests as liquefactive necrosis (Fig. 2.8).

Gangrenous necrosis is not a specific pattern of cell death, but the term is commonly used in clinical practice. It is usually applied to a limb, generally the lower leg, that has lost its blood supply and has undergone necrosis (typically coagulative necrosis) involving multiple tissue planes. When bacterial infection is superimposed, there is more liquefactive necrosis because of the actions of degradative enzymes in the bacteria and the attracted leukocytes (giving rise to so-called wet gangrene).

Caseous necrosis is encountered most often in foci of tuberculous infection (Chapter 8). The term caseous (cheeselike) is derived from the friable white appearance of the area of necrosis (Fig. 2.9). On microscopic examination, the necrotic area appears as a structureless collection of fragmented or lysed cells and amorphous granular debris enclosed within a distinctive inflammatory border; this appearance is characteristic of a focus of inflammation known as a granuloma (Chapter 3).

Fat necrosis refers to focal areas of fat destruction, typically resulting from release of activated pancreatic lipases into the substance of the pancreas and the peritoneal cavity. This occurs in the calamitous abdominal emergency known as acute pancreatitis (Chapter 19). In this disorder, pancreatic enzymes leak out of damaged acinar cells and liquefy the membranes of fat cells in the peritoneum, releasing triglyceride esters that are split by pancreatic lipases. Fatty acids are generated that combine with calcium to produce grossly visible chalky-white areas (fat saponification), which enable the surgeon and the pathologist to identify the underlying disorder (Fig. 2.10). On histologic examination, the necrotic areas contain the shadowy outlines of necrotic fat cells, basophilic calcium deposits, and an inflammatory reaction.

Fibrinoid necrosis is a special form of vascular damage usually seen in immune reactions involving blood vessels. It typically occurs when complexes of antigens and antibodies are deposited in the walls of arteries. Deposits of these immune complexes, together with plasma proteins that has leaked out of vessels, result in a bright pink and amorphous appearance in H&E stains called “fibrinoid” (fibrin-like) by pathologists (Fig. 2.11). The immunologically mediated vasculitis syndromes in which this type of vascular injury is seen are described in Chapter 11.

Ultimately, in the living patient most necrotic cells and their contents disappear due to enzymatic digestion and phagocytosis of the debris by leukocytes. If necrotic cells and cellular debris are not promptly destroyed and reabsorbed, they provide a nidus for the deposition of calcium salts and other minerals and thus tend to become calcified. This phenomenon, called dystrophic calcification, is considered later in the chapter.

Key Concepts

Cell Injury and Necrosis

• • Exposure of cells to stress or noxious agents causes cell injury that is reversible to a point but may progress to death of the cells, principally by necrosis.
• • Reversible cell injury: Characterized by cellular swelling, fatty change, plasma membrane blebbing and loss of microvilli, mitochondrial swelling, dilation of the ER, and eosinophilia (due to decreased cytoplasmic RNA)
• • Necrosis: A pathologic process in which cellular membranes are destroyed, enzymes and other constituents leak out, and local inflammation is induced to clear the damaged cells. Morphologic features are: eosinophilia; nuclear shrinkage, fragmentation, and dissolution; breakdown of plasma membrane and organellar membranes; abundant myelin figures; and leakage and enzymatic digestion of cellular contents
• • Patterns of tissue necrosis: Under different conditions, necrosis in tissues may assume specific patterns: coagulative, liquefactive, gangrenous, caseous, fat, and fibrinoid

Apoptosis

Apoptosis is a type 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’ genomic DNA and nuclear and cytoplasmic proteins. Apoptotic cells break up into plasma membrane–bound fragments, called apoptotic bodies, which contain portions of the cytoplasm and nucleus. While the plasma membrane remains intact, its surface components are altered so as to produce “find me” and “eat me” signals for phagocytes, discussed later. As a result, the dead cell and its fragments are rapidly devoured, before the contents leak out, and therefore apoptosis does not elicit an inflammatory reaction. Apoptosis was first recognized in 1972 by the distinctive morphologic appearance of membrane-bound fragments derived from cells, and named after the Greek designation for “falling off.” It was subsequently discovered in model organisms such as worms that certain cells undergo apoptosis at precise times during development. This phenomenon, termed programmed cell death, is controlled by the action of a small number of genes and is required for normal embyrogenesis. Thus, apoptosis is a unique mechanism of cell death, distinct from necrosis in many respects (see Fig. 2.4 and Table 2.1).

Morphologic and Biochemical Changes in Apoptosis

Before discussing underlying mechanisms, the morphologic and biochemical characteristics of apoptosis are described.

Morphology

The following morphologic features, some best seen with the electron microscope, characterize cells undergoing apoptosis (Fig. 2.12; see Fig. 2.4).

Cell shrinkage. Cell size is reduced, the cytoplasm is dense and eosinophilic (see Fig. 2.12A), and the organelles, although relatively normal, are more tightly packed. This contrasts with necrosis, in which an early feature is cell swelling, not shrinkage.

Chromatin condensation. This is the most characteristic feature of apoptosis. The chromatin aggregates peripherally, under the nuclear membrane, into dense masses of various shapes and sizes (see Fig. 2.12B). The nucleus itself may break up into two or more fragments.

Formation of cytoplasmic blebs and apoptotic bodies. The apoptotic cell first shows extensive surface membrane blebbing, which is followed by fragmentation of the dead cells into membrane-bound apoptotic bodies composed of cytoplasm and tightly packed organelles, with or without nuclear fragments (see Fig. 2.12C).

Phagocytosis of apoptotic cells or cell bodies, usually by macrophages. The apoptotic bodies are rapidly ingested by phagocytes and degraded by the phagocyte's lysosomal enzymes.

In H&E-stained tissue, the apoptotic cell appears as a round or oval mass of intensely eosinophilic cytoplasm with fragments of dense nuclear chromatin (see Fig. 2.12A). Because the cell shrinkage and formation of apoptotic bodies are rapid and the pieces are quickly cleared by phagocytes, considerable apoptosis may occur in tissues before it is apparent in histologic sections. The absence of an inflammatory response can also make it difficult to detect apoptosis by light microscopy.

Mechanisms of Apoptosis

Apoptosis results from the activation of enzymes called caspases (so named because they are proteases containing a cysteine in their active site and cleave proteins after aspartic residues). Like many proteases, caspases exist as inactive proenzymes and must undergo enzymatic cleavage to become active. The presence of active caspases is therefore a marker for cells undergoing apoptosis (see Fig. 2.12C). The process of apoptosis may be divided into an initiation phase, during which some caspases become catalytically active and unleash a cascade of other caspases, and an execution phase, during which the terminal caspases trigger cellular fragmentation. Regulation of these enzymes depends on a finely tuned balance between the 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 (Fig. 2.13). Although these pathways intersect, they are generally induced under different conditions, involve different initiating molecules, and serve distinct roles in physiology and disease.

The Mitochondrial (Intrinsic) Pathway of Apoptosis

The mitochondrial pathway is responsible for apoptosis in most physiologic and pathologic situations. It results from increased permeability of the mitochondrial outer membrane with consequent release of death-inducing (pro-apoptotic) molecules from the mitochondrial intermembrane space into the cytoplasm (Fig. 2.14). Mitochondria are organelles that contain remarkable proteins such as cytochrome c, a double-edged sword that is essential for producing the energy (e.g., ATP) that sustains cell viability, but that when released into the cytoplasm (an indication that the cell is not healthy) initiates the suicide program of apoptosis. The release of pro-apoptotic proteins such as cytochrome c is determined by the integrity of the outer mitochondrial membrane, which is tightly controlled by the BCL2 family of proteins. This family is named after BCL2, a gene that is frequently overexpressed due to chromosomal translocations and other aberrations in certain B cell lymphomas (Chapter 13). There are more than 20 members of the BCL family, which can be divided into three groups based on their pro-apoptotic or anti-apoptotic function and the BCL2 homology (BH) domains they possess.

• •  Anti-apoptotic. BCL2, BCL-X_L, and MCL1 are the principal members of this group; they possess four BH domains (called BH1-4). These proteins reside in the outer mitochondrial membrane as well as in the cytosol and ER membranes. By keeping the mitochondrial outer membrane impermeable, they prevent leakage of cytochrome c and other death-inducing proteins into the cytosol (see Fig. 2.14A).
• •  Pro-apoptotic. BAX and BAK are the two prototypic members of this group; they contain the first three BH domains (BH1-3). On activation, BAX and/or BAK oligomerize within the outer mitochondrial membrane and enhance its permeability. The precise mechanism by which BAX-BAK oligomers permeabilize membranes is not settled. According to one model illustrated in Fig. 2.14B, they form a channel in the outer mitochondrial membrane that allows cytochrome c leakage from the intermembranous space.
• •  Regulated apoptosis initiators. Members of this group, including BAD, BIM, BID, Puma, and Noxa, contain only one BH domain, the third of the four BH domains, and hence are sometimes called BH3-only proteins. The activity of BH3-only proteins is modulated by sensors of cellular stress and damage; when upregulated and activated, they can initiate apoptosis.

Growth factors and other survival signals stimulate the production of anti-apoptotic proteins such as BCL2, thus protecting cells from apoptosis. When cells are deprived of survival signals, suffer DNA damage, or develop ER stress due to the accumulation of misfolded proteins, BH3-only proteins are upregulated through increased transcription and/or post-translational modifications (e.g., phosphorylation). These BH3-only proteins in turn directly activate the two critical pro-apoptotic family members, BAX and BAK, which form oligomers that insert into the mitochondrial membrane and allow proteins from the inner mitochondrial membrane to leak out into the cytoplasm. BH3-only proteins may also bind to and block the function of BCL2 and BCL-X_L. At the same time, synthesis of BCL2 and BCL-X_L may decline because their transcription relies on survival signals. The net result of BAX-BAK activation coupled with loss of the protective functions of the anti-apoptotic BCL2 family members is the release into the cytoplasm of several mitochondrial proteins such as cytochrome c that can activate the caspase cascade (see Fig. 2.14).

Once released into the cytosol, cytochrome c binds to a protein called APAF-1 (apoptosis-activating factor-1), forming a multimeric structure called the apoptosome. This complex binds to caspase-9, the critical initiator caspase of the mitochondrial pathway, and promotes its autocatalytic cleavage, generating catalytically active forms of the enzyme. Active caspase-9 then triggers a cascade of caspase activation by cleaving and thereby activating other pro-caspases (such as caspase-3), which mediate the execution phase of apoptosis (discussed later). Other mitochondrial proteins with arcane names like Smac/DIABLO enter the cytoplasm, where they bind to and neutralize cytoplasmic proteins that function as physiologic inhibitors of apoptosis (IAPs). The normal function of the IAPs is to block the inappropriate activation of caspases, including executioners like caspase-3, and keep cells alive. Thus, IAP inhibition permits initiation of the caspase cascade.

The Extrinsic (Death Receptor–Initiated) Pathway of Apoptosis

This pathway is initiated by engagement of plasma membrane death receptors. Death receptors are members of the tumor necrosis factor (TNF) receptor family that contain a cytoplasmic domain involved in protein-protein interactions. This death domain is essential for delivering apoptotic signals. (Some TNF receptor family members do not contain cytoplasmic death domains; their function is to activate inflammatory cascades [Chapter 3], and their role in triggering apoptosis is much less established.) The best-known death receptors are the type 1 TNF receptor (TNFR1) and a related protein called Fas (CD95), but several others have been described. The mechanism of apoptosis induced by these death receptors is well illustrated by Fas, a death receptor expressed on many cell types (Fig. 2.15). The ligand for Fas is called Fas ligand (FasL). FasL is expressed on T cells that recognize self antigens (and functions to eliminate self-reactive lymphocytes that also express the receptor Fas upon recognition of self antigens) and on some CTLs that kill virus-infected and tumor cells. When FasL binds to Fas, three or more molecules of Fas are brought together, and their cytoplasmic death domains form a binding site for an adaptor protein called FADD (Fas-associated death domain). Once attached to this complex, FADD binds inactive caspase-8 (or caspase-10), bringing together multiple caspase molecules and leading to autocatalytic cleavage and generation of active caspase-8. In turn, active caspase-8 initiates the same executioner caspase sequence as in the mitochondrial pathway. This extrinsic apoptosis pathway can be inhibited by a protein called FLIP, which binds to pro-caspase-8, thereby blocking FADD binding, but cannot activate the caspase. Some viruses and normal cells produce FLIP as a mechanism to protect themselves from Fas-mediated apoptosis.

The extrinsic and intrinsic pathways of apoptosis are initiated in fundamentally different ways by distinct molecules, but there may be interconnections between them. For instance, in hepatocytes and pancreatic β cells, caspase-8 produced by Fas signaling cleaves and activates the BH3-only protein BID, which then feeds into the mitochondrial pathway. The combined activation of both pathways delivers a fatal blow to the cells.

The Execution Phase of Apoptosis

The intrinsic and extrinsic pathways converge to activate a caspase cascade that mediates the final phase of apoptosis. The intrinsic mitochondrial pathway activates the initiator caspase-9, whereas the extrinsic death receptor pathway activates caspase-8 and caspase-10. The active forms of these caspases trigger the rapid and sequential activation of the executioner caspases, such as caspase-3 and caspase-6, which then act on many cellular components. For instance, once activated these caspases cleave an inhibitor of a DNase, making the DNase enzymatically active and allowing DNA degradation to commence. Caspases also proteolyze structural components of the nuclear matrix and thus promote fragmentation of nuclei. Other steps in apoptosis are less well-defined. For instance, we do not know how membrane blebs and apoptotic bodies are formed.

Removal of Dead Cells

The formation of apoptotic bodies breaks cells up into “bite-sized” fragments that are edible for phagocytes. Apoptotic cells and their fragments also undergo several changes in their membranes that actively promote their phagocytosis so they are most often cleared before they lose membrane integrity and release their cellular contents. In healthy cells, phosphatidylserine is present on the inner leaflet of the plasma membrane, but in apoptotic cells this phospholipid “flips” out and is expressed on the outer layer of the membrane, where it is recognized by several macrophage receptors. Cells that are dying by apoptosis also secrete soluble factors that recruit phagocytes, and macrophages themselves may produce proteins that bind to apoptotic cells (but not live cells), leading to their engulfment. Apoptotic bodies may also become coated with natural antibodies and proteins of the complement system, notably C1q, which are recognized by phagocytes. Thus, numerous ligands induced on apoptotic cells serve as “eat me” signals and are recognized by receptors on phagocytes that bind and engulf these cells. This process of apoptotic cell phagocytosis is called efferocytosis; it is so efficient that dead cells disappear, often within minutes, without leaving a trace. In addition, production of pro-inflammatory cytokines is reduced in macrophages that have ingested apoptotic cells. Together with rapid clearance, this limits inflammatory reactions, even in the face of extensive apoptosis.

Key Concepts

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), which leads to leakage of pro-apoptotic proteins from mitochondrial membrane into the cytoplasm and subsequent caspase activation; can be inhibited by anti-apoptotic members of the BCL2 family, which are induced by survival signals including growth factors
  • • Death receptor (extrinsic) pathway eliminates self-reactive lymphocytes and is a mechanism of cell killing by cytotoxic T lymphocytes; is initiated by engagement of death receptors (members of the TNF receptor family). The responsible ligands can be soluble or expressed on the surface of adjacent cells

Other Mechanisms of Cell Death

• •  Necroptosis. As the name indicates, this form of cell death is a hybrid that shares aspects of both necrosis and apoptosis. Morphologically, and to some extent biochemically, it resembles necrosis, as both are characterized by loss of ATP, swelling of the cell and organelles, generation of reactive oxygen species (ROS), release of lysosomal enzymes, and ultimately rupture of the plasma membrane. Mechanistically, it is triggered by signal transduction pathways that culminate in cell death, a feature similar to apoptosis. Because of these overlapping features, necroptosis is sometimes called programmed necrosis to distinguish it from forms of necrosis driven passively by toxic or ischemic injury to the cell. In sharp contrast to apoptosis, the signals leading to necroptosis do not result in caspase activation, and hence it is also sometimes referred to as “caspase-independent” programmed cell death. The process of necroptosis starts in a manner similar to that of the extrinsic form of apoptosis, that is, by ligation of a receptor by its ligand. Ligation of TNFR1 is the most widely studied model of necroptosis, but many other signals, including ligation of Fas and yet to be identified sensors of viral DNA and RNA, can also trigger necroptosis. Since TNF can cause both apoptosis and necroptosis, the mechanisms underlying these effects of TNF are especially illustrative (Fig. 2.16).

  
  

  
  Although the entire set of signaling molecules and their interactions are not known, necroptosis involves two kinases called receptor-interacting protein kinase 1 and 3 (RIPK1 and RIPK3). As indicated in Fig. 2.16, ligation of TNFR1 recruits these kinases into a multiprotein complex, and RIPK3 phosphorylates a cytoplasmic protein called MLKL. In response to its phosphorylation, MLKL monomers assemble into oligomers, translocate from the cytosol to the plasma membrane, and cause the plasma membrane disruption that is characteristic of necrosis. This explains the morphologic similarity of necroptosis with necrosis initiated by other injuries.
  Necroptosis is postulated to be an important death pathway both in physiologic and pathologic conditions. For example, physiologic necroptosis occurs during the formation of the mammalian bone growth plate. In pathologic states, it is associated with cell death in steatohepatitis, acute pancreatitis, ischemia-reperfusion injury, and neurodegenerative diseases such as Parkinson disease. Necroptosis also acts as a backup mechanism in host defense against certain viruses that encode caspase inhibitors (e.g., cytomegalovirus).
• •  Pyroptosis is a form of apoptosis that is accompanied by the release of the fever-inducing cytokine IL-1 (pyro refers to fever). Microbial products that enter infected cells are recognized by cytoplasmic innate immune receptors and can activate the multiprotein complex called the inflammasome (Chapter 6). The function of the inflammasome is to activate caspase-1 (also known as interleukin-1β–converting enzyme), which cleaves a precursor form of interleukin-1 (IL-1) and releases its biologically active form. IL-1 is a mediator of many aspects of inflammation, including leukocyte recruitment and fever (Chapter 3). Caspase-1 and the closely related caspases-4 and -5 also induce death of the cells. Unlike classical apoptosis, this pathway of cell death is characterized by release of inflammatory mediators. Pyroptosis is thought to be the mechanism by which some microbes cause the death of infected cells and at the same time trigger local inflammation.
• •  Ferroptosis. Only discovered in 2012, ferroptosis is a distinct form of cell death that is triggered when excessive intracellular levels of iron or reactive oxygen species overwhelm the glutathione-dependent antioxidant defenses (discussed later) to cause unchecked membrane lipid peroxidation. The widespread peroxidation of lipids disrupts many aspects of membrane function, including fluidity, lipid-protein interactions, ion and nutrient transport, and signaling pathways. The overall effect is the loss of plasma membrane permeability, which ultimately leads to cell death resembling necrosis. The process is, however, regulated by specific signals (unlike necrosis) and can be prevented by reducing iron levels (hence its name). Ultrastructurally, the most prominent features are the loss of mitochondrial cristae and ruptured outer mitochondrial membrane. While its role in normal development and physiology remain controversial, ferroptosis has been linked to cell death in a variety of human pathologies, including cancer, neurodegenerative diseases, and stroke.

Autophagy

Autophagy is a process in which a cell eats its own contents (Greek: auto, self; phagy, eating). It involves the delivery of cytoplasmic materials to the lysosome for degradation. Autophagy is an evolutionarily conserved survival mechanism whereby, in states of nutrient deprivation, starved cells live by cannibalizing themselves and recycling the digested contents. Autophagy is implicated in many physiologic states (e.g., aging and exercise) and pathologic processes. It proceeds through several steps (Fig. 2.17):

• • Nucleation and formation of an isolation membrane, also called a phagophore; the isolation membrane is believed to be derived from the ER, though other membrane sources such as the plasma membrane and mitochondria may contribute
• • Formation of a vesicle, called the autophagosome, from the isolation membrane, inside which intracellular organelles and cytosolic structures are sequestered
• • Maturation of the autophagosome by fusion with lysosomes, to deliver digestive enzymes that degrade the contents of the autophagosome

In recent years, more than a dozen “autophagy-related genes” called Atgs have been identified whose products are required for the creation of the autophagosome. Environmental cues like nutrient deprivation or depletion of growth factors activate an initiation complex of four proteins that promotes the hierarchical recruitment of Atgs to nucleate the initiation membrane. The initiation membrane elongates further, surrounds and captures its cytosolic cargo, and closes to form the autophagosome. The elongation and closure of the initiation membrane require the coordinated action of two ubiquitin-like conjugation systems that result in the covalent linkage of the lipid phosphatidylethanolamine (PE) to microtubule-associated protein light chain 3 (LC3). PE-lipidated LC3 is increased during autophagy, and it is therefore a useful marker for identifying cells in which autophagy is occurring. The newly formed autophagosome fuses with lysosomes to form an autophagolysosome. In the terminal step, the inner membrane and enclosed cytosolic cargoes are degraded by lysosomal enzymes. There is increasing evidence that autophagy is not a random process that engulfs cytosolic contents indiscriminately. Rather, the loading of cargo into the autophagosome is selective, and one of the functions of the lipidated LC3 is to target protein aggregates and effete organelles.

Autophagy functions as a survival mechanism under various stress conditions, maintaining the integrity of cells by recycling essential metabolites and clearing intracellular debris. It is therefore prominent in atrophic cells exposed to severe nutrient deprivation. Autophagy is also involved in the turnover of organelles like the ER, mitochondria, and lysosomes and the clearance of intracellular aggregates that accumulate during aging, stress, and various disease states. Autophagy can trigger cell death if it is inadequate to cope with the stressor. This pathway of cell death is distinct from necrosis and apoptosis, but the mechanism is unknown. Furthermore, it is not clear whether cell death is caused by autophagy or by the stress that triggered autophagy. Nevertheless, autophagic vacuolization often precedes or accompanies cell death.

There is accumulating evidence that autophagy plays a role in human diseases, including the following:

• •  Cancer: Autophagy can both promote cancer growth and act as a defense against cancers. This is an area of active investigation, as discussed in Chapter 7.
• •  Neurodegenerative disorders: Many neurodegenerative disorders are associated with dysregulation of autophagy. Alzheimer disease is characterized by impaired autophagosome maturation, and in mouse models of the disease genetic defects in autophagy accelerate neurodegeneration. In Huntington disease, mutant huntingtin impairs autophagy.
• •  Infectious diseases: Many pathogens are degraded by autophagy; these include mycobacteria, Shigella spp., and HSV-1. This is one way by which microbial proteins are digested and delivered to antigen presentation pathways. Macrophage-specific deletion of Atg5 increases susceptibility to tuberculosis.
• •  Inflammatory bowel diseases: Genome-wide association studies have linked both Crohn disease and ulcerative colitis to single-nucleotide polymorphisms (SNPs) in the autophagy-related gene ATG16L1. How these polymorphisms promote intestinal inflammation is not known.

General Mechanisms of Cell Injury and Intracellular Targets of Injurious Stimuli

Cell injury results from abnormalities in one or more essential cellular components (Fig. 2.18). The principal targets of injurious stimuli are mitochondria, cell membranes, the machinery of protein synthesis and secretion, and DNA. The consequences of injury of each of these cellular components are distinct but overlapping.

Mitochondrial Damage

Mitochondria are critical players in all pathways leading to cell injury and death. This should be expected because mitochondria supply life-sustaining energy by producing ATP but are also targets of many injurious stimuli. Thus, in many ways, they are the arbiters of life and death of cells. Mitochondria can be damaged by increases of cytosolic Ca²⁺, ROS (discussed later), and oxygen deprivation, which makes them sensitive to virtually all types of injurious stimuli, including hypoxia and toxins. In addition, mutations in mitochondrial genes are the cause of some inherited diseases (Chapter 5).

There are three major consequences of mitochondrial damage.

• •  ATP depletion. Decreased ATP synthesis and ATP depletion are frequently associated with both hypoxic and chemical (toxic) injury (Fig. 2.19). ATP is produced in two ways. The major pathway in mammalian cells, particularly those that are nondividing (e.g., brain and liver), is oxidative phosphorylation of adenosine diphosphate in a reaction that results in reduction of oxygen by the mitochondrial electron transport system. The second is the glycolytic pathway, which can generate ATP, albeit in far smaller amounts, in the absence of oxygen using glucose derived either from body fluids or from the hydrolysis of glycogen. In addition to mitochondrial damage, the major causes of ATP depletion are reduced supply of oxygen and nutrients (because of ischemia and hypoxia), and the actions of some toxins (e.g., cyanide). Mitochondrial damage often results in the formation of a high-conductance channel in the mitochondrial membrane, called the mitochondrial permeability transition pore (see Fig. 2.19). The opening of this conductance channel leads to the loss of mitochondrial membrane potential, resulting in failure of oxidative phosphorylation and progressive ATP depletion that culminates in necrosis.

  
  

  
  High-energy phosphate in the form of ATP is required for virtually all synthetic and degradative processes within the cell. These include membrane transport, protein synthesis, lipogenesis, and the deacylation-reacylation reactions necessary for phospholipid turnover. Hence, depletion of ATP to 5% to 10% of normal levels has widespread effects on many critical cellular systems.
  • • The activity of the plasma membrane energy-dependent sodium pump (Na⁺,K⁺-ATPase) is reduced (Chapter 1). Failure of this active transport system causes sodium to enter and accumulate inside cells and potassium concentrations to fall. The net solute gain results in osmotically driven water accumulation that leads to cell swelling and ER dilation.
  • •  Cellular energy metabolism is altered. If the supply of oxygen to cells is reduced, as in ischemia, oxidative phosphorylation ceases, resulting in a decrease in cellular ATP and associated increase in adenosine monophosphate. These changes stimulate phosphorylase and phosphofructokinase activities, leading to increased rates of glycogenolysis and glycolysis, respectively, in an effort to maintain energy supplies by generating ATP through metabolism of glucose derived from glycogen. As a consequence, glycogen stores are rapidly depleted. Glycolysis under anaerobic conditions results in the accumulation of lactic acid and inorganic phosphates from the hydrolysis of phosphate esters. This reduces the intracellular pH, resulting in decreased activity of many cytosolic enzymes.
  • • With prolonged or worsening depletion of ATP, structural disruption of the protein synthetic apparatus occurs, manifest as detachment of ribosomes from the rough ER and dissociation of polysomes, with a consequent reduction in protein synthesis. There may also be increased protein misfolding, with injurious effects that are discussed later.
  • • Ultimately, there is irreversible damage to mitochondrial and lysosomal membranes, and the cell undergoes necrosis.
• • Incomplete oxidative phosphorylation also leads to the formation of ROS, which have many deleterious effects, described later.
• • As discussed earlier, leakage of mitochondrial proteins due to channel formation by pro-apoptotic BAX and BAK is the initial step in apoptosis by the intrinsic pathway. This action of BAX and BAK is specific to mitochondrial membranes only and leads to damage of other organelles indirectly.

Membrane Damage

Early loss of selective membrane permeability, leading ultimately to overt membrane damage, is a consistent feature of most forms of cell injury (except apoptosis). Membrane damage may affect the integrity and functions of all cellular membranes. In ischemic cells, membrane defects may be the result of ATP depletion and calcium-mediated activation of phospholipases. The plasma membrane can also be damaged directly by bacterial toxins, viral proteins, lytic complement components, and a variety of physical and chemical agents. Several biochemical mechanisms may contribute to membrane damage (Fig. 2.20):

• •  ROS. Oxygen free radicals cause injury to cell membranes by lipid peroxidation, discussed later.
• •  Decreased phospholipid synthesis. The production of phospholipids in cells may be reduced as a consequence of defective mitochondrial function or hypoxia, both of which decrease ATP production and thus affect energy-dependent biosynthetic pathways. Decreased phospholipid synthesis may affect all cellular membranes, including those of mitochondria.
• •  Increased phospholipid breakdown. Severe cell injury is associated with increased degradation of membrane phospholipids, probably due to activation of calcium-dependent phospholipases by increased cytosolic and mitochondrial Ca2⁺. Phospholipid breakdown leads to the accumulation of lipid breakdown products, including unesterified free fatty acids, acyl carnitine, and lysophospholipids, which have a detergent effect on membranes. They may also either insert into the lipid bilayer of the membrane or exchange with membrane phospholipids, potentially causing changes in permeability and electrophysiologic alterations.
• •  Cytoskeletal abnormalities. Cytoskeletal filaments serve as anchors connecting the plasma membrane to the cell interior, and proteases activated by cytosolic Ca²⁺ may damage these tethers. When exacerbated by cell swelling, particularly in myocardial cells, this damage leads to detachment of the cell membrane from the cytoskeleton, rendering it susceptible to stretching and rupture.

Damage to different cellular membranes has diverse effects on cells.

• •  Mitochondrial membrane damage. As discussed earlier, damage to mitochondrial membranes results in opening of the mitochondrial permeability transition pore, leading to decreased ATP generation and release of proteins that trigger apoptotic death.
• •  Plasma membrane damage. Plasma membrane damage results in loss of osmotic balance and influx of fluids and ions, as well as loss of cellular contents. The cells may also leak metabolites (e.g., glycolytic intermediates) that are vital for the reconstitution of ATP, thus further depleting energy stores.
• •  Injury to lysosomal membranes results in leakage of their enzymes into the cytoplasm and activation of acid hydrolases in the acidic intracellular pH of the injured cell. These lysosomal hydrolases include RNases, DNases, proteases, phosphatases, and glucosidases, which degrade RNA, DNA, proteins, phosphoproteins, and glycogen, respectively, and push cells into necrosis.

Damage to DNA

Damage to nuclear DNA activates sensors that trigger p53-dependent pathways (Chapter 7). DNA damage may be caused by exposure to radiation, chemotherapeutic (anticancer) drugs, and ROS, or may occur spontaneously as a part of aging, due largely to deamination of cytosine residues to uracil residues. DNA damage activates p53, which arrests cells in the G1 phase of the cell cycle and activates DNA repair mechanisms. If these mechanisms fail to correct the DNA damage, p53 triggers apoptosis by the mitochondrial pathway. Thus, the cell chooses to die rather than survive with abnormal DNA that has the potential to induce malignant transformation. Predictably, mutations in p53 that interfere with its ability to arrest cell cycling or to induce apoptosis are associated with numerous cancers (Chapter 7).

In addition to damage to various organelles, some biochemical alterations are involved in many situations that lead to cell injury. Two of these general pathways are discussed next.

Oxidative Stress: Accumulation of Oxygen-Derived Free Radicals

ROS are a type of oxygen-derived free radical whose role in cell injury is well established. ROS are produced normally in cells during mitochondrial respiration and energy generation, but they are degraded and removed by intracellular ROS scavengers. These defense systems allow cells to maintain a steady state in which free radicals may be present at low concentrations but do not cause damage. Increased production or decreased scavenging of ROS may lead to an excess of free radicals, a condition called oxidative stress. Oxidative stress has been implicated in a wide variety of pathologic processes, including cell injury, cancer, aging, and some degenerative diseases, such as Alzheimer disease. ROS are also produced in large amounts by activated leukocytes, particularly neutrophils and macrophages, during inflammatory reactions aimed at destroying microbes and cleaning up dead cells and other unwanted substances (Chapter 3).

The following section discusses the generation and removal of ROS, and how they contribute to cell injury. The properties of some of the most important free radicals are summarized in Table 2.2.

Table 2.2

Properties of the Principal Free Radicals Involved in Cell Injury

Properties		H2O2	˙OH	ONOO−
Mechanisms of production	Incomplete reduction of O2 during oxidative phosphorylation; by phagocyte oxidase in leukocytes	Generated by SOD from and by oxidases in peroxisomes	Generated from H2O by hydrolysis (e.g., by radiation); from H2O2 by Fenton reaction; from	Produced by interaction of and NO generated by NO synthase in many cell types (endothelial cells, leukocytes, neurons, others)
Mechanisms of inactivation	Conversion to H2O2 and O2 by SOD	Conversion to H2O and O2 by catalase (peroxisomes), glutathione peroxidase (cytosol, mitochondria)	Conversion to H2O by glutathione peroxidase	Conversion to HNO2 by peroxiredoxins (cytosol, mitochondria)
Pathologic effects	Stimulates production of degradative enzymes in leukocytes and other cells; may directly damage lipids, proteins, DNA; acts close to site of production	Can be converted to OH and OCl−, which destroy microbes and cells; can act distant from site of production	Most reactive oxygen-derived free radical; principal ROS responsible for damaging lipids, proteins, and DNA	Damages lipids, proteins, DNA

HNO₂, Nitrite; H₂O₂, hydrogen peroxide; NO, nitric oxide; , superoxide anion; OCl⁻, hypochlorite; OH, hydroxyl radical; ONOO⁻, peroxynitrite; ROS, reactive oxygen species; SOD, superoxide dismutase.

Generation of Free Radicals

Free radicals may be generated within cells in several ways (Fig. 2.21):

• •  The reduction-oxidation reactions that occur during normal metabolic processes. As a part of normal respiration, molecular O₂ is reduced by the transfer of four electrons to H₂ to generate two water molecules. This conversion is catalyzed by oxidative enzymes in the ER, cytosol, mitochondria, peroxisomes, and lysosomes. During this process, small amounts of partially reduced intermediates are produced in which different numbers of electrons have been transferred from O₂; these include superoxide anion (, one electron), hydrogen peroxide (H₂O₂, two electrons), and hydroxyl radicals (˙OH, three electrons).
• •  Absorption of radiant energy (e.g., ultraviolet light, x-rays). For example, ionizing radiation can hydrolyze water into ˙OH and hydrogen (H) free radicals.
• • Rapid bursts of ROS are produced in activated leukocytes during inflammation. This occurs in a precisely controlled reaction carried out by a plasma membrane multiprotein complex that uses NADPH oxidase for the redox reaction (Chapter 3). In addition, some intracellular oxidases (e.g., xanthine oxidase) generate . Defects in leukocytic superoxide production lead to chronic granulomatous disease (Chapter 6).
• •  Enzymatic metabolism of exogenous chemicals or drugs can generate free radicals that are not ROS but have similar effects (e.g., CCl₄ can generate ˙CCl₃, described later in the chapter).
• •  Transition metals such as iron and copper donate or accept free electrons during intracellular reactions and catalyze free radical formation, as in the Fenton reaction (H₂O₂ + Fe²⁺ → Fe³⁺ + ˙OH + OH⁻). Because most of the intracellular free iron is in the ferric (Fe³⁺) state, it must be reduced to the ferrous (Fe²⁺) form to participate in the Fenton reaction. This reduction can be enhanced by , and thus sources of iron and may cooperate in oxidative cell damage.
• •  Nitric oxide (NO), an important chemical mediator generated by endothelial cells, macrophages, neurons, and other cell types (Chapter 3), can act as a free radical and may also be converted to highly reactive peroxynitrite anion (ONOO⁻) as well as NO₂ and NO₃ ⁻.

Disturbance in Calcium Homeostasis

Calcium ions normally serve as second messengers in several signaling pathways, but if released into the cytoplasm of cells in excessive amounts, are also an important source of cell injury. In keeping with this, calcium depletion protects cultured cells from injury induced by a variety of harmful stimuli. Cytosolic free Ca²⁺ is normally maintained at very low concentrations (~0.1?µmol) compared with extracellular levels of 1.3?mmol, and most intracellular Ca²⁺ is sequestered in mitochondria and the ER. Ischemia and certain toxins cause an excessive increase in cytosolic Ca²⁺, initially because of release from intracellular stores, and later due to increased influx across the plasma membrane (Fig. 2.22). Excessive intracellular Ca²⁺ may cause cell injury by several mechanisms, although the significance of these mechanisms in cell injury in vivo is not established.

• • The accumulation of Ca²⁺ in mitochondria results in opening of the mitochondrial permeability transition pore and, as described earlier, failure of ATP generation.
• • Increased cytosolic Ca²⁺ activates a number of enzymes with potentially deleterious effects on cells. These enzymes include phospholipases (which cause membrane damage), proteases (which break down both membrane and cytoskeletal proteins), endonucleases (which are responsible for DNA and chromatin fragmentation), and ATPases (thereby hastening ATP depletion).

Endoplasmic Reticulum Stress: the Unfolded Protein Response

The accumulation of misfolded proteins in the ER can stress adaptive mechanisms and trigger apoptosis. Chaperones in the ER control the proper folding of newly synthesized proteins, and misfolded polypeptides are shuttled into the cytoplasm where they are ubiquitinated and targeted for proteolysis in proteasomes (Chapter 1). If, however, unfolded or misfolded proteins accumulate in the ER, they trigger a number of alterations that are collectively called the unfolded protein response. The unfolded protein response activates signaling pathways that increase the production of chaperones, enhance proteasomal degradation of abnormal proteins, and slow protein translation, thus reducing the load of misfolded proteins in the cell (Fig. 2.23). However, if this cytoprotective response is unable to cope with the accumulation of misfolded proteins, the cell activates caspases and induces apoptosis. This process is called ER stress. Intracellular accumulation of misfolded proteins may be caused by an increased rate of misfolding or a reduction in the cell's ability to repair or eliminate them. Increased misfolding may be a consequence of deleterious mutations or decreased capacity to correct misfolded proteins, as occurs in aging. Protein misfolding may also be increased in viral infections when proteins encoded by the viral genome are synthesized in such large quantities that they overwhelm the quality control system that normally ensures proper protein folding. Increased demand for secretory proteins such as insulin in insulin-resistant states, and changes in intracellular pH and redox state are other stressors that result in misfolded protein accumulation. Protein misfolding is thought to be the causative cellular abnormality in several neurodegenerative diseases (Chapter 28). Given that many “foldases” require ATP to function, deprivation of glucose and oxygen, as in ischemia and hypoxia, also may increase the burden of misfolded proteins. Diseases caused by misfolded proteins are listed in Table 2.3.

Hypoxia and Ischemia

Mechanisms of Ischemic Cell Injury

The sequence of events following hypoxia or ischemia reflects many of the biochemical alterations in cell injury, summarized here in Fig. 2.24 and shown earlier in Figs. 2.5 and 2.6. As intracellular oxygen tension falls, oxidative phosphorylation fails and ATP generation decreases. The consequences of ATP depletion were described earlier in the Mitochondrial Damage section. In brief, loss of ATP results initially in reversible cell injury (cell and organelle swelling) and later in cell death by necrosis.

Mammalian cells have developed protective responses to deal with hypoxic stress. The best defined of these is induction of a transcription factor called hypoxia-inducible factor-1 (HIF-1), which promotes new blood vessel formation, stimulates cell survival pathways, and enhances glycolysis. Several promising investigational compounds that promote HIF-1 signaling are being developed. Nevertheless, there are still no reliable therapeutic approaches for reducing the injurious consequences of ischemia in clinical situations. The strategy that is perhaps the most useful in ischemic (and traumatic) brain and spinal cord injury is the transient induction of hypothermia (lowering the core body temperature to 92°F). This treatment reduces the metabolic demands of the stressed cells, decreases cell swelling, suppresses the formation of free radicals, and inhibits the host inflammatory response. All of these may contribute to decreased cell and tissue injury.

Ischemia-Reperfusion Injury

Restoration of blood flow to ischemic tissues can promote recovery of cells if they are reversibly injured but can also paradoxically exacerbate cell injury and cause cell death. As a consequence, reperfused tissues may sustain loss of viable cells in addition to those that are irreversibly damaged by the ischemia. This process, called ischemia-reperfusion injury, is clinically important because it contributes to tissue damage during myocardial and cerebral infarction following therapies that restore blood flow (Chapters 12 and 28).

How does reperfusion injury occur? The likely answer is that new damaging processes are set in motion during reperfusion, causing the death of cells that might have recovered otherwise. Several mechanisms have been proposed:

• •  Oxidative stress. New damage may be initiated during reoxygenation by increased generation of reactive oxygen and nitrogen species. These free radicals may be produced in reperfused tissue as a result of incomplete reduction of oxygen in leukocytes, and in damaged endothelial cells and parenchymal cells. Compromise of cellular antioxidant defense mechanisms during ischemia may sensitize cells to free radical damage.
• •  Intracellular calcium overload. As mentioned earlier, intracellular and mitochondrial calcium overload begins during acute ischemia; it is exacerbated during reperfusion due to influx of calcium resulting from cell membrane damage and ROS-mediated injury to sarcoplasmic reticulum. Calcium overload favors opening of the mitochondrial permeability transition pore with resultant ATP depletion. This, in turn, causes further cell injury.
• •  Inflammation. Ischemic injury is associated with inflammation as a result of “danger signals” released from dead cells, cytokines secreted by resident immune cells such as macrophages, and increased expression of adhesion molecules by hypoxic parenchymal and endothelial cells, all of which act to recruit circulating neutrophils to reperfused tissue. The inflammation causes additional tissue injury (Chapter 3). The importance of neutrophil influx in reperfusion injury has been demonstrated experimentally by the salutary effects of treatment with antibodies that block cytokines or adhesion molecules and thereby reduce neutrophil extravasation.
• • Activation of the complement system may contribute to ischemia-reperfusion injury. For unknown reasons, some IgM antibodies have a propensity to deposit in ischemic tissues. When blood flow is resumed, complement proteins bind to the deposited antibodies, are activated, and exacerbate cell injury and inflammation.

Chemical (Toxic) Injury

Chemical injury remains a frequent problem in clinical medicine and is a major limitation to drug therapy. Because many drugs are metabolized in the liver, this organ is a major target of drug toxicity. In fact, toxic liver injury is often the reason for terminating the therapeutic use or development of a drug. The mechanisms by which chemicals, certain drugs, and toxins produce injury are described in greater detail in Chapter 9 in the discussion of environmental diseases. Here the major pathways of chemically induced injury with selected examples are described.

Chemicals induce cell injury by one of two general mechanisms:

• •  Direct toxicity. Some chemicals injure cells directly by combining with critical molecular components. For example, in mercuric chloride poisoning, mercury binds to the sulfhydryl groups of cell membrane proteins, causing increased membrane permeability and inhibition of ion transport. In such instances, the greatest damage is usually to the cells that use, absorb, excrete, or concentrate the chemicals—in the case of mercuric chloride, the cells of the gastrointestinal tract and kidney (Chapter 9). Cyanide poisons mitochondrial cytochrome oxidase and thus inhibits oxidative phosphorylation. Many antineoplastic chemotherapeutic agents and antibiotics also induce cell damage by direct cytotoxic effects.
• •  Conversion to toxic metabolites. Most toxic chemicals are not biologically active in their native form but must be converted to reactive toxic metabolites, which then act on target molecules. This modification is usually accomplished by the cytochrome P-450 mixed-function oxidases in the smooth ER of the liver and other organs. The toxic metabolites cause membrane damage and cell injury mainly by formation of free radicals and subsequent lipid peroxidation; direct covalent binding to membrane proteins and lipids may also contribute. For instance, CCl₄, which was once widely used in the dry cleaning industry, is converted by cytochrome P-450 to the highly reactive free radical ˙CCl₃, which causes lipid peroxidation and damages many cellular structures. The analgesic drug acetaminophen is also converted to a toxic product during detoxification in the liver, leading to cell injury. These and other examples of chemical injury are described in Chapter 9.

Hypertrophy

• •  Pathologic hypertrophy. The striated muscle cells in the heart and skeletal muscles have only a limited capacity for division, and respond to increased metabolic demands mainly by undergoing hypertrophy. The most common stimulus for hypertrophy of skeletal and cardiac muscle is increased workload. In both tissue types, muscle cells respond by synthesizing more protein and increasing the number of myofilaments per cell. This in turn increases the amount of force each myocyte can generate and thus the strength and work capacity of the muscle as a whole. A classic example of pathologic hypertrophy is enlargement of the heart in response to pressure overload, usually resulting from either hypertension or valvular disease (see Fig. 2.2). Initially, cardiac hypertrophy improves function, but over time this adaptation often fails, setting the stage for heart failure and other significant forms of heart disease (Chapter 12).
• •  Physiologic hypertrophy. The massive physiologic growth of the uterus during pregnancy is a good example of hormone-induced enlargement of an organ that results mainly from hypertrophy of smooth muscle fibers (Fig. 2.25). Uterine hypertrophy during pregnancy is stimulated by estrogenic hormone signaling through estrogen receptors that eventually result in increased synthesis of smooth muscle proteins and an increased cell size. The bulging muscles of bodybuilders engaged in “pumping iron” result from enlargement of individual skeletal muscle fibers in response to increased demand.

  
  

Mechanisms of Hypertrophy

Hypertrophy is a result of increased cellular protein production. Much of our understanding of hypertrophy is based on studies of the heart. There is great interest in defining the molecular basis of myocardial hypertrophy because beyond a certain point, it becomes maladaptive. Hypertrophy results from the action of growth factors and direct effects on cellular proteins (Fig. 2.26):

• • Mechanical sensors in the cell detect the increased load.
• • These sensors activate a complex downstream web of signaling pathways, including the phosphoinositide 3-kinase (PI3K)/AKT pathway (postulated to be most important in physiologic, e.g., exercise-induced, hypertrophy) and G-protein–coupled receptor–initiated pathways (activated by many growth factors and vasoactive agents, and thought to be more important in pathologic hypertrophy).
• • Some of the signaling pathways stimulate increased production of growth factors (e.g. TGF-β, insulin-like growth factor 1 [IGF1], fibroblast growth factor) and vasoactive agents (e.g., α-adrenergic agonists, endothelin-1, and angiotensin II).
• • These and other pathways activate transcription factors, including GATA4, nuclear factor of activated T cells (NFAT), and myocyte enhancer factor 2 (MEF2), which increase the expression of genes that encode muscle proteins.

Cardiac hypertrophy is also associated with a switch in gene expression from genes that encode adult-type contractile proteins to genes that encode functionally distinct fetal isoforms of the same proteins. For example, the α isoform of myosin heavy chain is replaced by the β isoform, which has a slower, more energetically economical contraction. Other proteins that are altered in hypertrophic myocardial cells are the products of genes that participate in the cellular response to stress. For example, cardiac hypertrophy is associated with increased atrial natriuretic factor gene expression. Atrial natriuretic factor is a peptide hormone that causes salt secretion by the kidney, decreases blood volume and pressure, and therefore serves to reduce hemodynamic load.

Whatever the exact cause and mechanism of cardiac hypertrophy, it eventually reaches a limit beyond which enlargement of muscle mass is no longer able to cope with the increased burden. At this stage, several regressive changes occur in the myocardial fibers, of which the most important are degradation and loss of myofibrillar contractile elements. In extreme cases, myocyte death can occur. The net result of these changes is cardiac failure, a sequence of events that illustrates how an adaptation to stress can progress to functionally significant cell injury if the stress is not relieved.

Hyperplasia

• •  Physiologic hyperplasia. Physiologic hyperplasia due to the action of hormones or growth factors occurs when there is a need to increase functional capacity of hormone sensitive organs, or when there is need for compensatory increase after damage or resection. Hormonal hyperplasia is well illustrated by the proliferation of the glandular epithelium of the female breast at puberty and during pregnancy, usually accompanied by enlargement (hypertrophy) of the glandular epithelial cells. The classic illustration of compensatory hyperplasia comes from the study of liver regeneration. In individuals who donate one lobe of the liver for transplantation, the remaining cells proliferate so that the organ soon grows back to its original size. Experimental models of partial hepatectomy have been very useful for defining the mechanisms that stimulate regeneration of the liver (Chapter 3). The bone marrow is also remarkable in its capacity to undergo rapid hyperplasia in response to a deficiency of mature blood cells. For example, in the setting of an acute bleeding or premature breakdown of red blood cells (hemolysis), feedback loops involving the growth factor erythropoietin are activated that stimulate the growth of red blood cell progenitors, allowing red blood cell production to increase as much as eightfold. The regulation of hematopoiesis is discussed further in Chapter 13.
• •  Pathologic hyperplasia. Most forms of pathologic hyperplasia are caused by excessive or inappropriate actions of hormones or growth factors acting on target cells. Endometrial hyperplasia is an example of abnormal hormone-induced hyperplasia. Normally, after a menstrual period there is a rapid burst of proliferative activity in the endometrium that is stimulated by pituitary hormones and ovarian estrogen. It is brought to a halt by the rising levels of progesterone, usually about 10 to 14 days before the end of the menstrual period. In some instances, however, the balance between estrogen and progesterone is disturbed, resulting in absolute or relative increases in the amount of estrogen, with consequent hyperplasia of the endometrial glands. This form of pathologic hyperplasia is a common cause of abnormal uterine bleeding. Benign prostatic hyperplasia is another common example of pathologic hyperplasia, in this case as a response to hormonal stimulation by androgens. Although these forms of pathologic hyperplasias are abnormal, the process remains controlled and the hyperplasia can either regress or stabilize if the hormonal stimulation is eliminated. As is discussed in Chapter 7, the increased cell division associated with hyperplasia elevates the risk of acquiring genetic aberrations that drive unrestrained proliferation and cancer. Thus, although hyperplasia is distinct from cancer, pathologic hyperplasia constitutes a fertile soil in which cancerous proliferations may eventually arise. For instance, patients with hyperplasia of the endometrium are at increased risk for developing endometrial cancer (Chapter 22).
  Hyperplasia is a characteristic response to certain viral infections, such as papillomaviruses, which cause skin warts and several mucosal lesions composed of masses of hyperplastic epithelium. Here, the viruses make factors that interfere with host proteins that regulate cell proliferation. Like other forms of hyperplasia, some of these virally induced proliferations are also precursors to cancer (Chapter 7).

Atrophy

• •  Decreased workload (disuse atrophy). When a fractured bone is immobilized in a plaster cast or when a patient is restricted to complete bed rest, skeletal muscle atrophy rapidly ensues. The initial decrease in cell size is reversible once activity is resumed. With more prolonged disuse, skeletal muscle fibers decrease in number (due to apoptosis) as well as in size; muscle atrophy can be accompanied by increased bone resorption, leading to osteoporosis of disuse.
• •  Loss of innervation (denervation atrophy). The normal metabolism and function of skeletal muscle are dependent on its nerve supply. Damage to the nerves leads to atrophy of the muscle fibers supplied by those nerves (Chapter 27).
• •  Diminished blood supply. A gradual decrease in blood supply (chronic ischemia) to a tissue as a result of slowly developing arterial occlusive disease results in tissue atrophy. In late adult life, the brain may undergo progressive atrophy, mainly because of reduced blood supply as a result of atherosclerosis (Fig. 2.27). This is called senile atrophy.

  
  

• •  Inadequate nutrition. Profound protein-calorie malnutrition (marasmus) is associated with the utilization of skeletal muscle proteins as a source of energy after other reserves such as adipose stores have been depleted. This results in marked muscle wasting (cachexia; Chapter 9). Cachexia is also seen in patients with chronic inflammatory diseases and cancer. In some cachectic states, chronic overproduction of the inflammatory cytokine TNF is thought to be responsible for appetite suppression and lipid depletion, culminating in muscle atrophy.
• •  Loss of endocrine stimulation. Many hormone-responsive tissues, such as the breast and reproductive organs, are dependent on endocrine stimulation for normal metabolism and function. The loss of estrogen stimulation after menopause results in atrophy of the endometrium, vaginal epithelium, and breast. Similarly, the prostrate atrophies following chemical or surgical castration (e.g., for treatment of prostate cancer).
• •  Pressure. Tissue compression for any length of time can cause atrophy. An enlarging benign tumor can cause atrophy in the surrounding uninvolved tissues. Atrophy in this setting is probably the result of ischemic changes caused by compromise of the blood supply by the pressure exerted by the expanding mass.

The fundamental cellular changes associated with atrophy are similar in all of these settings. The initial response is a decrease in cell size and organelles, which may reduce the metabolic needs of the cell sufficiently to permit its survival. In atrophic muscle, the cells contain fewer mitochondria and myofilaments and a reduced amount of rough ER. By bringing into balance the cell's metabolic demands and the lower levels of blood supply, nutrition, or trophic stimulation, a new equilibrium is achieved. Early in the process, atrophic cells and tissues have diminished function, but cell death is minimal. However, atrophy caused by gradually reduced blood supply may progress to the point at which cells are irreversibly injured and die, often by apoptosis. Cell death by apoptosis also contributes to the atrophy of endocrine organs after hormone withdrawal.

Metaplasia

The most common epithelial metaplasia is columnar to squamous (Fig. 2.28), as occurs in the respiratory tract in response to chronic irritation. In the habitual cigarette smoker, the normal ciliated columnar epithelial cells of the trachea and bronchi are often replaced by stratified squamous epithelial cells. Vitamin A (retinoic acid) deficiency can also induce squamous metaplasia in the respiratory epithelium and in the cornea, the latter with highly deleterious effects on vision (Chapter 9). Stones in the excretory ducts of the salivary glands, pancreas, or bile ducts, which are normally lined by secretory columnar epithelium, may also lead to squamous metaplasia. In all these instances, the more rugged stratified squamous epithelium is able to survive under circumstances in which the more fragile specialized columnar epithelium might have succumbed. However, the change to metaplastic squamous cells comes with a price. In the respiratory tract, for example, although the epithelial lining becomes more durable, important mechanisms of protection against infection—mucus secretion and the ciliary action of the columnar epithelium—are lost. Thus, epithelial metaplasia, in most circumstances, represents an undesirable change. Moreover, the influences that predispose to metaplasia, if persistent, can initiate malignant transformation in metaplastic epithelium. The development of squamous cell carcinoma in areas of the lungs where the normal columnar epithelium has been replaced by squamous epithelium is one example.

Metaplasia from squamous to columnar type may also occur, as in Barrett esophagus, in which the esophageal squamous epithelium is replaced by intestinal-like columnar cells under the influence of refluxed gastric acid. As might be expected, the cancers that arise in these areas are typically glandular (adenocarcinomas) (Chapter 17).

Connective tissue metaplasia is the formation of cartilage, bone, or adipose cells (mesenchymal tissues) in tissues that normally do not contain these elements. For example, bone formation in muscle, designated myositis ossificans, occasionally occurs after intramuscular hemorrhage. This type of metaplasia is less clearly seen as an adaptive response, and may be a result of cell or tissue injury. Unlike epithelial metaplasia, this type of metaplasia is not associated with increased cancer risk.

Lipids

All major classes of lipids can accumulate in cells: triglycerides, cholesterol/cholesterol esters, and phospholipids. Phospholipids are components of the myelin figures found in necrotic cells. In addition, abnormal complexes of lipids and carbohydrates accumulate in the lysosomal storage diseases (Chapter 5). Triglyceride and cholesterol accumulations are discussed here.

Steatosis (Fatty Change)

The terms steatosis and fatty change describe abnormal accumulations of triglycerides within parenchymal cells. Fatty change is often seen in the liver because it is the major organ involved in fat metabolism (Fig. 2.30), but it also occurs in the heart, muscle, and kidney. The causes of steatosis include toxins, protein malnutrition, diabetes mellitus, obesity, and anoxia. In higher-income nations, the most common causes of significant fatty change in the liver (fatty liver) are alcohol abuse and nonalcoholic fatty liver disease, which is often associated with diabetes and obesity. Fatty liver is discussed in more detail in Chapter 18.

Proteins

Intracellular accumulations of proteins usually appear as rounded, eosinophilic droplets, vacuoles, or aggregates in the cytoplasm. By electron microscopy, they can be amorphous, fibrillar, or crystalline in appearance. In some disorders, such as certain forms of amyloidosis, abnormal proteins deposit primarily in extracellular spaces (Chapter 6).

Excesses of proteins within the cells sufficient to cause morphologically visible accumulation have diverse causes.

• •  Reabsorption droplets in proximal renal tubules are seen in renal diseases associated with protein loss in the urine (proteinuria). In the kidney, small amounts of protein filtered through the glomerulus are normally reabsorbed by pinocytosis in the proximal tubule. In disorders with heavy protein leakage across the glomerular filter, there is increased reabsorption of the protein into vesicles, and the protein appears as pink hyaline droplets within the cytoplasm of the tubular cell (Fig. 2.32). The process is reversible; if the proteinuria diminishes, the protein droplets are metabolized and disappear.

  
  

• • The proteins that accumulate may be normal secreted proteins that are produced in excessive amounts and accumulate within the ER, as occurs in certain plasma cells engaged in active synthesis of immunoglobulins. The ER becomes hugely distended, producing large, homogeneous eosinophilic inclusions called Russell bodies.
• •  Defective intracellular transport and secretion of critical proteins. In α₁-antitrypsin deficiency, mutations in the protein significantly slow folding, resulting in the buildup of partially folded intermediates, which aggregate in the ER of the hepatocyte and are not secreted. The resultant deficiency of the circulating enzyme where it is needed in the lung causes emphysema (Chapter 15). In many of these protein-folding diseases, the pathology results not only from loss of protein function but also ER stress caused by the misfolded proteins, which initiates the unfolded protein response and culminates in cell death by apoptosis (discussed earlier).
• •  Accumulation of cytoskeletal proteins. There are several types of cytoskeletal proteins, including microtubules (20 to 25?nm in diameter), thin actin filaments (6 to 8?nm), thick myosin filaments (15?nm), and intermediate filaments (10?nm). Intermediate filaments, which provide a flexible intracellular scaffold that organizes the cytoplasm and resists forces applied to the cell, are divided into five classes: keratin filaments (characteristic of epithelial cells), neurofilaments (neurons), desmin filaments (muscle cells), vimentin filaments (connective tissue cells), and glial filaments (astrocytes). Accumulations of keratin filaments and neurofilaments are associated with certain types of cell injury. Alcoholic hyaline is an eosinophilic cytoplasmic inclusion in liver cells that is characteristic of alcoholic liver disease and is composed predominantly of keratin intermediate filaments (Chapter 18). The neurofibrillary tangle found in the brain in Alzheimer disease contains neurofilaments and other proteins (Chapter 28).
• •  Aggregation of abnormal proteins. Abnormal or misfolded proteins may deposit in tissues and interfere with normal functions. The deposits can be intracellular, extracellular, or both, and the aggregates may either directly or indirectly cause the pathologic changes. Certain forms of amyloidosis (Chapter 6) fall in this category of diseases. These disorders are sometimes called proteinopathies or protein-aggregation diseases.

Hyaline Change

Glycogen

Glycogen accumulates within select cells in a group of related genetic disorders that are collectively referred to as the glycogen storage diseases, or glycogenoses (Chapter 5). In these diseases, enzymatic defects in the synthesis or breakdown of glycogen result in massive accumulation, causing cell injury and cell death.

Pigments

Endogenous Pigments

Lipofuscin is an insoluble pigment, also known as lipochrome or wear-and-tear pigment. Lipofuscin is composed of polymers of lipids and phospholipids in complex with protein, suggesting that it is derived through lipid peroxidation of polyunsaturated lipids of intracellular membranes. Lipofuscin is not injurious to the cell or its functions. Its importance lies in its being a telltale sign of free radical injury and lipid peroxidation. The term is derived from the Latin (fuscus, brown), referring to brown lipid. In tissue sections, it appears as a yellow-brown, finely granular cytoplasmic, often perinuclear, pigment (Fig. 2.33). It is seen in cells undergoing slow, regressive changes and is particularly prominent in the liver and heart of aging patients or patients with severe malnutrition and cancer cachexia.

Melanin, derived from the Greek (melas, black), is an endogenous, brown-black, pigment formed when the enzyme tyrosinase catalyzes the oxidation of tyrosine to dihydroxyphenylalanine in melanocytes. It is discussed further in Chapter 25. For practical purposes, melanin is the only endogenous brown-black pigment. The only other that could be considered in this category is homogentisic acid, a black pigment that occurs in patients with alkaptonuria, a rare metabolic disease. Here the pigment is deposited in the skin, connective tissue, and cartilage, and the pigmentation is known as ochronosis.

Hemosiderin, a hemoglobin-derived, golden yellow-to-brown, granular, or crystalline pigment is one of the major storage forms of iron. Iron metabolism and hemosiderin are considered in detail in Chapters 14 and 18. Iron is normally carried by a specific transport protein called transferrin. In cells, it is stored in association with a protein, apoferritin, to form ferritin micelles. Ferritin is a constituent of most cell types. When there is a local or systemic excess of iron, ferritin forms hemosiderin granules, which are easily seen with the light microscope. Hemosiderin pigment represents aggregates of ferritin micelles. Under normal conditions, small amounts of hemosiderin can be seen in the mononuclear phagocytes of the bone marrow, spleen, and liver, which are responsible for recycling of iron derived from hemoglobin during the breakdown of effete red blood cells.

Local or systemic excesses of iron cause hemosiderin to accumulate within cells. Local excesses result from hemorrhages in tissues. The best example of localized hemosiderosis is the common bruise. Extravasated red blood cells at the site of injury are phagocytosed over several days by macrophages, which break down the hemoglobin and recover the iron. After removal of iron, the heme moiety is converted first to biliverdin (“green bile”) and then to bilirubin (“red bile”). In parallel, the iron released from heme is incorporated into ferritin and eventually hemosiderin. These conversions account for the often dramatic play of colors seen in a healing bruise, which typically changes from red-blue to green-blue to golden-yellow before it is resolved.

When there is systemic iron overload, hemosiderin may be deposited in many organs and tissues, a condition called hemosiderosis. The main causes of hemosiderosis are (1) increased absorption of dietary iron due to an inborn error of metabolism called hemochromatosis (Chapter 18), (2) hemolytic anemias, in which excessive lysis of red blood cells leads to release of abnormal quantities of iron (Chapter 14), and (3) repeated blood transfusions, because transfused red blood cells constitute an exogenous iron load.

Dystrophic Calcification

Dystrophic calcification is encountered in areas of necrosis, whether they are of coagulative, caseous, or liquefactive type, and in foci of enzymatic necrosis of fat. Calcification is almost always present in the atheromas of advanced atherosclerosis. It also commonly develops in aging or damaged heart valves, further hampering their function (Fig. 2.34). Whatever the site of deposition, the calcium salts appear macroscopically as fine, white granules or clumps, often felt as gritty deposits. Sometimes a tuberculous lymph node is virtually converted to stone.

Although dystrophic calcification may simply be a telltale sign of previous cell injury, it is often a cause of organ dysfunction. Such is the case in calcific valvular disease and atherosclerosis, as will become clear in further discussion of these diseases (Chapters 11 and 12). Serum calcium is normal in dystrophic calcification.

Metastatic Calcification

Metastatic calcification may occur in normal tissues whenever there is hypercalcemia. Hypercalcemia also accentuates dystrophic calcification. There are four principal causes of hypercalcemia: (1) increased secretion of parathyroid hormone (PTH) with subsequent bone resorption, as in hyperparathyroidism due to parathyroid tumors, and ectopic secretion of PTH-related protein by malignant tumors (Chapter 7); (2) resorption of bone tissue, secondary to primary tumors of bone marrow (e.g., multiple myeloma, leukemia) or diffuse skeletal metastasis (e.g., breast cancer), accelerated bone turnover (e.g., Paget disease), or immobilization; (3) vitamin D–related disorders, including vitamin D intoxication, sarcoidosis (in which macrophages activate a vitamin D precursor), and idiopathic hypercalcemia of infancy (Williams syndrome), characterized by abnormal sensitivity to vitamin D; and (4) renal failure, which causes retention of phosphate, leading to secondary hyperparathyroidism. Less common causes include aluminum intoxication, which occurs in patients on chronic renal dialysis, and milk-alkali syndrome, which is due to excessive ingestion of calcium and absorbable antacids such as milk or calcium carbonate.

Metastatic calcification may occur widely throughout the body but principally affects the interstitial tissues of the gastric mucosa, kidneys, lungs, systemic arteries, and pulmonary veins. Although quite different in location, all of these tissues excrete acid and therefore have an internal alkaline compartment that predisposes them to metastatic calcification. In all of these sites, the calcium salts morphologically resemble those described in dystrophic calcification. Thus, they may occur as noncrystalline amorphous deposits or, at other times, as hydroxyapatite crystals.

Usually the mineral salts cause no clinical dysfunction, but on occasion massive involvement of the lungs produces remarkable x-ray images and respiratory compromise. Massive deposits in the kidney (nephrocalcinosis) may in time cause renal damage (Chapter 20).