UNIT I General Pathology 1 Cellular Adaptations, Cell Injury, and Cell Death

Introduction to Pathology
Pathology is literally the study (logos) of suffering (pathos). More specifically, it is a bridging discipline involving both basic science and clinical practice and is devoted to the study of the structural and functional changes in cells, tissues, and organs that underlie disease. By the use of olecular, icrobiologic, i unologic, and orphologic techni!ues, pathology atte pts to e"plain the #hys and #herefores of the signs and sy pto s anifested by patients #hile providing a sound foundation for rational clinical care and therapy. $raditionally, the study of pathology is divided into general pathology and special, or syste ic, pathology. $he for er is concerned #ith the basic reactions of cells and tissues to abnor al sti uli that underlie all diseases. $he latter e"a ines the specific responses of speciali%ed organs and tissues to ore or less #ell&defined sti uli. In this boo', #e first cover the principles of general pathology and then proceed to specific disease processes as they affect particular organs or syste s. $he four aspects of a disease process that for the core of pathology are its cause (etiology), the echanis s of its develop ent (pathogenesis), the structural alterations induced in the cells and organs of the body (morphologic changes), and the functional conse!uences of the orphologic changes (clinical significance). Etiology or Cause. $he concept that certain abnor al sy pto s or diseases are (caused( is as ancient as recorded history. )or the Arcadians *+,-- BC., if so eone beca e ill, it #as the patient/s o#n fault *for having sinned. or the a'ings of outside agents, such as bad s ells, cold, evil spirits, or gods.1 In odern ter s, there are t#o ajor classes of etiologic factors0 intrinsic or genetic, and ac!uired *e.g., infectious, nutritional, che ical, physical.. $he concept, ho#ever, of one etiologic agent for one disease & developed fro the study of infections or single&gene disorders & is no longer sufficient. 1enetic factors are clearly involved in so e of the co on environ entally induced aladies, such as atherosclerosis and cancer, and the environ ent ay also have profound influences on certain genetic diseases. 2no#ledge or discovery of the pri ary cause re ains the bac'bone on #hich a diagnosis can be ade, a disease understood, or a treat ent developed. Pathogenesis. Pathogenesis refers to the se!uence of events in the response of cells or tissues to the etiologic agent, fro the initial sti ulus to the ulti ate e"pression of the disease. $he study of pathogenesis re ains one of the ain do ains of pathology. 3ven #hen the initial infectious or olecular cause is 'no#n, it is any steps re oved fro the e"pression of the disease. )or e"a ple, to understand cystic fibrosis is to 'no# not only the defective gene and gene product, but also the bioche ical, i unologic, and orphologic events leading to the for ation of cysts and fibrosis in the lung, pancreas, and other organs. Indeed, as #e shall see throughout the boo', the olecular revolution has already identified utant genes underlying a great nu ber of diseases, and the entire hu an geno e has been apped. 4evertheless, the functions of the encoded proteins and ho# utations induce disease are often still obscure. Because of technologic advances, it is beco ing increasingly feasible to lin' specific olecular abnor alities to disease anifestations and to use this 'no#ledge to design ne# therapeutic approaches. )or these reasons, the study of pathogenesis has never been ore e"citing scientifically or ore relevant to edicine. Morphologic Changes. $he orphologic changes refer to the structural alterations in cells or tissues that are either characteristic of the disease or diagnostic of the etiologic process. $he

practice of diagnostic pathology is devoted to identifying the nature and progression of disease by studying orphologic changes in tissues and che ical alterations in patients. More recently, the li itations of orphology for diagnosing diseases have beco e increasingly evident, and the field of diagnostic pathology has e"panded to enco pass olecular biologic and i unologic approaches for analy%ing disease states. 4o#here is this ore stri'ing than in the study of tu ors & breast cancers and tu ors of ly phocytes that loo' orphologically identical ay have #idely different courses, therapeutic responses, and prognosis. Molecular analysis by techni!ues such as D4A icroarrays has begun to reveal genetic differences that bear on the behavior of the tu ors. Increasingly, such techni!ues are being used to e"tend and even supplant traditional orphologic ethods. Functional Derangements and Clinical Manifestations. $he nature of the orphologic changes and their distribution in different organs or tissues influence nor al function and deter ine the clinical features *sy pto s and signs., course, and prognosis of the disease. 5irtually all for s of organ injury start #ith olecular or structural alterations in cells, a concept first put forth in the nineteenth century by 6udolf 5ircho#, 'no#n as the father of odern pathology. 7e therefore begin our consideration of pathology #ith the study of the origins, olecular echanis s, and structural changes of cell injury. 8et different cells in tissues constantly interact #ith each other, and an elaborate syste of extracellular matrix is necessary for the integrity of organs. Cell&cell and cell& atri" interactions contribute significantly to the response to injury, leading collectively to tissue and organ injury, #hich are as i portant as cell injury in defining the orphologic and clinical patterns of disease.

Cellular

daptations of Gro!th and Differentiation

Cells respond to increased de and and e"ternal sti ulation by hyperplasia or hypertrophy, and they respond to reduced supply of nutrients and gro#th factors by atrophy. In so e situations, cells change fro one type to another, a process called metaplasia. $here are nu erous olecular echanis s for cellular adaptations. 9o e adaptations are induced by direct sti ulation of cells by factors produced by the responding cells the selves or by other cells in the environ ent. :thers are due to activation of various cell surface receptors and do#nstrea signaling path#ays. Adaptations ay be associated #ith the induction of ne# protein synthesis by the target cells, as in the response of uscle cells to increased physical de and, and the induction of cellular proliferation, as in responses of the endo etriu to estrogens. Adaptations can also involve a s#itch by cells fro producing one type of proteins to another or ar'edly overproducing one protein; such is the case in cells producing various types of collagens and e"tracellular atri" proteins in chronic infla ation and fibrosis *Chapters + and <..

Figure "#" $tages in the cellular response to stress and in%urious stimuli.

Ta&le "#". Cellular 'esponses to In%ury Nature and $e(erity of In%urious $timulus ltered physiologic stimuli) Cellular 'esponse Cellular adaptations)

= Increased de and, increased trophic sti ulation *e.g. = >yperplasia, hypertrophy gro#th factors, hor ones. = Decreased nutrients, sti ulation = Chronic irritation *che ical or physical. = Atrophy = Metaplasia

'educed o*ygen supply+ chemical in%ury+ micro&ial Cell in%ury) infection = Acute and self&li ited = Progessive and severe *including D4A da age. = Acute reversible injury = Irreversible injury ? cell death 4ecrosis Apoptosis = Mild chronic injury = 9ubcellular alterations in various organelles Intracellular accumulations+ calcifications Cellular aging

Meta&olic alterations, genetic or ac-uired Prolonged life span !ith cumulati(e su&lethal in%ury

)igure 1&+ $he relationships bet#een nor al, adapted, reversibly injured, and dead yocardial cells. $he cellular adaptation depicted here is hypertrophy, and the type of cell death is ische ic necrosis. In reversibly injured yocardiu , generally effects are only functional, #ithout any readily apparent gross or even icroscopic changes. In the e"a ple of yocardial hypertrophy, the left ventricular #all is ore than + c in thic'ness *nor al is 1 to 1., c .. In the speci en sho#ing necrosis, the trans ural light area in the posterolateral left ventricle represents an acute yocardial infarction. All three transverse sections have been stained #ith triphenyltetra%oliu chloride, an en%y e substrate that colors viable yocardiu agenta. )ailure to stain is due to en%y e lea'age after cell death.

HYPERPLASIA >yperplasia is an increase in the nu ber of cells in an organ or tissue, usually resulting in increased volu e of the organ or tissue. Although hyperplasia and hypertrophy are t#o distinct processes, fre!uently both occur together, and they ay be triggered by the sa e e"ternal sti ulus. )or instance, hor one&induced gro#th in the uterus involves both increased nu bers of s ooth uscle and epithelial cells and the enlarge ent of these cells. >yperplasia ta'es place if the cellular population is capable of synthesi%ing D4A, thus per itting itotic division; by contrast, hypertrophy involves cell enlarge ent #ithout cell division. >yperplasia can be physiologic or pathologic. Physiologic Hyperplasia Physiologic hyperplasia can be divided into0 *1. hormonal hyperplasia, #hich increases the functional capacity of a tissue #hen needed, and *+. compensatory hyperplasia, #hich increases tissue ass after da age or partial resection. >or onal hyperplasia is best e"e plified by the proliferation of the glandular epitheliu of the fe ale breast at puberty and during pregnancy and the physiologic hyperplasia that occurs in the pregnant uterus. $he classical illustration of co pensatory hyperplasia co es fro the yth of Pro etheus, #hich sho#s that the ancient 1ree's recogni%ed the capacity of the liver to regenerate. As punish ent for having stolen the secret of fire fro the gods, Pro etheus #as chained to a ountain, and his liver #as devoured daily by a vulture, only to regenerate ane# every night. 1 $he e"peri ental odel of partial hepatecto y has been especially useful in e"a ining the echanis s that sti ulate proliferation of residual liver cells and regeneration of the liver *Chapter <.. 9i ilar echanis s are li'ely involved in other situations #hen re aining tissue gro#s to a'e up for partial tissue loss *e.g., after unilateral nephrecto y, #hen the re aining 'idney undergoes co pensatory hyperplasia..

Mechanisms of .yperplasia. >yperplasia is generally caused by increased local production of gro#th factors, increased levels of gro#th factor receptors on the responding cells, or activation of particular intracellular signaling path#ays. All these changes lead to production of transcription factors that turn on any cellular genes, including genes encoding gro#th factors, receptors for gro#th factors, and cell cycle regulators, and the net result is cellular proliferation. + In hor onal hyperplasia, the hor ones ay the selves act as gro#th factors and trigger the transcription of various cellular genes. $he source of gro#th factors in co pensatory hyperplasia and the sti uli for the production of these gro#th factors are less #ell defined. $he increase in tissue ass after so e types of cell loss is achieved not only by proliferation of the re aining cells but also by the develop ent of ne# cells fro stem cells.<,@ )or instance, in the liver, intrahepatic ste cells do not play a ajor role in the hyperplasia that occurs after hepatecto y but they ay participate in regeneration after certain for s of liver injury, such as chronic hepatitis, in #hich the proliferative capacity of hepatocytes is co pro ised. 6ecent data fro clinical observations and e"peri ental studies have de onstrated that the bone arro# contains ste cells that ay be able to give rise to any types of differentiated, speciali%ed cell types, including so e liver cells. , $hese observations highlight the plasticity of adult ste cells and raise the potential of repopulating da aged tissues #ith bone arro#&derived ste cells. 7e #ill return to a discussion of ste cells, their biology, and their clinical relevance in Chapter <. Pathologic Hyperplasia Most for s of pathologic hyperplasia are caused by e"cessive hor onal sti ulation or gro#th factors acting on target cells. 3ndo etrial hyperplasia is an e"a ple of abnor al hor one& induced hyperplasia. After a nor al enstrual period, there is a rapid burst of proliferative activity that is sti ulated by pituitary hor ones and ovarian estrogen. It is brought to a halt by the rising levels of progesterone , usually about 1- to 1@ days before the anticipated enstrual period. In so e instances, ho#ever, the balance bet#een estrogen and progesterone is disturbed. $his results in absolute or relative increases in the a ount of estrogen, #ith conse!uent hyperplasia of the endo etrial glands. $his for of hyperplasia is a co on cause of abnor al enstrual bleeding. Benign prostatic hyperplasia is another co on e"a ple of pathologic hyperplasia induced by responses to hor ones, in this case, androgens. Although these for s of hyperplasia are abnor al, the process re ains controlled, because the hyperplasia regresses if the hor onal sti ulation is eli inated. As is discussed in Chapter A, it is this response to nor al regulatory control echanis s that distinguishes benign pathologic hyperplasias fro cancer, in #hich the gro#th control echanis s beco e defective. Pathologic hyperplasia, however, constitutes a fertile soil in which cancerous proliferation may eventually arise . $hus, patients #ith hyperplasia of the endo etriu are at increased ris' for developing endo etrial cancer *Chapter ++.. >yperplasia is also an i portant response of connective tissue cells in #ound healing, in #hich proliferating fibro&blasts and blood vessels aid in repair *Chapter <.. Bnder these circu stances, gro#th factors are responsible for the hyperplasia. 9ti ulation by gro#th factors is also involved in the hyperplasia that is associated #ith certain viral infections, such as papillo aviruses, #hich cause s'in #arts and a nu ber of ucosal lesions co posed of asses of hyperplastic epitheliu .

HYPERTROPHY Hypertrophy refers to an increase in the size of cells, resulting in an increase in the size of the organ. $hus, the hypertrophied organ has no ne# cells, just larger cells. $he increased si%e of the cells is due not to cellular s#elling but to the synthesis of ore structural co ponents. As entioned above, cells capable of division ay respond to stress by undergoing both hyperplasia

and hypertrophy, #hereas in nondividing cells *e.g., yocardial fibers., hypertrophy occurs. 4uclei in hypertrophied cells ay have a higher D4A content than in nor al cells, probably because the cells arrest in the cell cycle #ithout undergoing itosis. >ypertrophy can be physiologic or pathologic and is caused by increased functional de and or by specific hor onal sti ulation. $he striated uscle cells in both the heart and the s'eletal uscles are capable of tre endous hypertrophy, perhaps because they cannot ade!uately adapt to increased etabolic de ands by itotic division and production of ore cells to share the #or'. $he ost co on sti ulus for hypertrophy of uscle is increased #or'load. )or e"a ple, the bulging uscles of bodybuilders engaged in (pu ping iron( result fro an increase in si%e of the individual uscle fibers in response to increased de and. $he #or'load is thus shared by a greater ass of cellular co ponents, and each uscle fiber is spared e"cess #or' and so escapes injury. $he enlarged uscle cell achieves a ne# e!uilibriu , per itting it to function at a higher level of activity. In the heart, the sti ulus for hypertrophy is usually chronic hemodynamic overload, resulting fro either hypertension or faulty valves. 9ynthesis of ore proteins and fila ents occurs, achieving a balance bet#een the de and and the cell/s functional capacity. $he greater nu ber of yofila ents per cell per its an increased #or'load #ith a level of etabolic activity per unit volu e of cell not different fro that borne by the nor al cell. $he assive physiologic gro#th of the uterus during pregnancy is a good e"a ple of hor one& induced increase in the si%e of an organ that results fro both hypertrophy and hyperplasia *)ig. 1& <A.. $he cellular hypertrophy is sti ulated by estrogenic hor ones acting on s ooth uscle estrogen receptors, eventually resulting in increased synthesis of s ooth uscle proteins and an increase in cell si%e *)ig. 1&< .. 9i ilarly, prolactin and estrogen cause hypertrophy of the breasts during lactation. $hese are e"a ples of physiologic hypertrophy induced by hor onal sti ulation. Although the traditional vie# of cardiac and s'eletal uscle is that these tissues are incapable of proliferation and, therefore, their enlarge ent is entirely a result of hypertrophy, recent data suggest that even these cell types are capable of li ited proliferation as #ell as repopulation fro precursors.C $his vie# e phasi%es the concept, entioned earlier, that hyperplasia and hypertrophy often occur conco itantly during the responses of tissues and organs to increased stress and cell loss.

)igure 1&< Physiologic hypertrophy of the uterus during pregnancy. A, 1ross appearance of a nor al uterus (right) and a gravid uterus *re oved for postpartu bleeding. (left). , 9 all spindle&shaped uterine s ooth uscle cells fro a nor al uterus (left) co pared #ith large

plu p cells in gravid uterus (right).

Mechanisms of .ypertrophy. Much of our understanding of hypertrophy is based on studies of the heart. $he echanis s of cardiac uscle hypertrophy involve any signal transduction path#ays, leading to the induction of a nu ber of genes, #hich in turn sti ulate synthesis of nu erous cellular proteins *)ig. 1&@..A,D $he genes that are induced during hypertrophy include those encoding transcription factors *such as c& fos, c&jun.; gro#th factors *$1)&E, insulin&li'e gro#th factor&1 FI1)&1G, fibroblast gro#th factor.; and vasoactive agents *H&adrenergic agonists, endothelin&1, and angiotensin II.. $hese factors are discussed in detail in Chapter <. $here ay also be a s#itch of contractile proteins fro adult to fetal or neonatal for s. )or e"a ple, during uscle hypertrophy, the H& yosin heavy chain is replaced by the E for of the yosin heavy chain, #hich leads to decreased yosin adenosine triphosphatase *A$Pase. activity and a slo#er, ore energetically econo ical contraction. In addition, so e genes that are e"pressed only during early develop ent are re&e"pressed in hypertrophic cells, and the products of these genes participate in the cellular response to stress. )or e"a ple, in the e bryonic heart, the gene for atrial natriuretic factor *A4). is e"pressed in both the atriu and the ventricle. After birth, ventricular e"pression of the gene is do#n&regulated. Cardiac hypertrophy, ho#ever, is associated #ith reinduction of A4) gene e"pression.I A4) is a peptide hor one that causes salt secretion by the 'idney, decreases blood volu e and pressure, and therefore serves to reduce he odyna ic load.

)igure 1&@ Changes in the e"pression of selected genes and proteins during hypertrophy.

yocardial

7hat are the triggers for hypertrophy and for these changes in gene e"pressionJ In the heart, there are at least t#o groups of signals0 mechanical triggers, such as stretch, and trophic triggers, such as polypeptide gro#th factors *I1)&1. and vasoactive agents *angiotensin II, H&adrenergic agonists.. Current odels suggest that gro#th factors or vasoactive agents produced by cardiac non uscle cells or by yocytes the selves in response to he odyna ic stress sti ulate the e"pression of various genes, leading to yocyte hypertrophy. $he si%e of cells is regulated by nutrients and environ ental cues and involves several signal transduction path#ays that are being unraveled.17hatever the e"act echanis of cardiac hypertrophy, it eventually reaches a li it beyond #hich enlarge ent of uscle ass is no longer able to co pensate for the increased burden, and

cardiac failure ensues. At this stage, a nu ber of degenerative changes occur in the yocardial fibers, of #hich the ost i portant are lysis and loss of yofibrillar contractile ele ents. Myocyte death can occur by either apoptosis or necrosis. 11 $he li iting factors for continued hypertrophy and the causes of the cardiac dysfunction are poorly understood; they ay be due to li itation of the vascular supply to the enlarged fibers, di inished o"idative capabilities of itochondria, alterations in protein synthesis and degradation, or cytos'eletal alterations. ATROPHY !hrin"age in the size of the cell #y loss of cell su#stance is "nown as atrophy . It represents a for of adaptive response and ay cul inate in cell death. 7hen a sufficient nu ber of cells are involved, the entire tissue or organ di inishes in si%e, or beco es atrophic. Atrophy can be physiologic or pathologic. Physiologic atrophy is co on during early develop ent. 9o e e bryonic structures, such as the notochord and thyroglossal duct, undergo atrophy during fetal develop ent. $he uterus decreases in si%e shortly after parturition, and this is a for of physiologic atrophy. Pathologic atrophy depends on the underlying cause and can be local or generali%ed. $he co on causes of atrophy are the follo#ing0 Decreased workload (atrophy of dis se!" 7hen a bro'en li b is i obili%ed in a plaster cast or #hen a patient is restricted to co plete bed rest, s'eletal uscle atrophy rapidly ensues. $he initial rapid decrease in cell si%e is reversible once activity is resu ed. 7ith ore prolonged disuse, s'eletal uscle fibers decrease in nu ber as #ell as in si%e; this atrophy can be acco panied by increased bone resorption, leading to osteoporosis of disuse. Loss of i##er$atio# (de#er$atio# atrophy!" 4or al function of s'eletal uscle is dependent on its nerve supply. Da age to the nerves leads to rapid atrophy of the uscle fibers supplied by those nerves *Chapter +A.. Di%i#ished &lood s pply. A decrease in blood supply *ische ia. to a tissue as a result of arterial occlusive disease results in atrophy of tissue o#ing to progressive cell loss. In late adult life, the brain undergoes progressive atrophy, presu ably as atherosclerosis narro#s its blood supply *)ig. 1&,.. I#ade' ate # tritio#. Profound protein&calorie alnutrition * aras us. is associated #ith the use of s'eletal uscle as a source of energy after other reserves such as adipose stores have been depleted. $his results in ar'ed uscle #asting (cachexia). Cache"ia is also seen in patients #ith chronic infla atory diseases and cancer. In the for er, chronic overproduction of the infla atory cyto'ine tu or necrosis factor *$4). is thought to be responsible for appetite suppression and uscle atrophy. Loss of e#docri#e sti% latio#" Many endocrine glands, the breast, and the reproductive organs are dependent on endocrine sti ulation for nor al etabolis and function. $he loss of estrogen sti ulation after enopause results in physiologic atrophy of the endo etriu , vaginal epitheliu , and breast. Agi#g (se#ile atrophy!" $he aging process is associated #ith cell loss, typically seen in tissues containing per anent cells, particularly the brain and heart. Press re" $issue co pression for any length of ti e can cause atrophy. An enlarging benign tu or can cause atrophy in the surrounding co pressed tissues. Atrophy in this setting is probably the result of ische ic changes caused by co pro ise of the blood supply to those tissues by the e"panding ass.

)igure 1&, A, Atrophy of the brain in an D+&year&old ale #ith atherosclerotic disease. Atrophy of the brain is due to aging and reduced blood supply. $he eninges have been stripped. () 4or al brain of a <C&year&old ale. 4ote that loss of brain substance narro#s the gyri and #idens the sulci.

$he funda ental cellular changes associated #ith atrophy are identical in all of these settings, representing a retreat by the cells to a s aller si%e at #hich survival is still possible. Atrophy results fro a reduction in the structural co ponents of the cell. In atrophic uscle, the cells contain fe#er itochondria and yofila ents and a reduced a ount of endoplas ic reticulu . By bringing into balance cell volu e and lo#er levels of blood supply, nutrition, or trophic sti ulation, a ne# e!uilibriu is achieved. Although atrophic cells may have diminished function, they are not dead. >o#ever, atrophy ay progress to the point at #hich cells are injured and die. In ische ic tissues, if the blood supply is inade!uate even to aintain the life of shrun'en cells, injury and cell death ay supervene. )urther ore, apoptosis ay be induced by the sa e signals that cause atrophy and thus ay contribute to loss of organ ass. )or e"a ple, apoptosis contributes to the regression of endocrine organs after hor one #ithdra#al. Mechanisms of trophy. $he bioche ical echanis s responsible for atrophy are inco pletely understood but are li'ely to affect the balance bet#een protein synthesis and degradation. Increased protein degradation probably plays a 'ey role in atrophy. Ma alian cells contain ultiple proteolytic syste s that serve distinct functions. $ysosomes contain acid hydrolases *e.g., cathepsins. and other en%y es that degrade endocytosed proteins fro the e"tracellular environ ent and the cell surface as #ell as so e cellular co ponents. $he u#i%uitin&proteasome pathway is responsible for the degradation of any cytosolic and nuclear proteins. 1+ Proteins to be degraded by this process are first conjugated to ubi!uitin and then degraded #ithin a large cytoplas ic proteolytic organelle called the proteasome. $his path#ay is thought to be responsible for the accelerated proteolysis seen in a variety of catabolic conditions, including cancer cache"ia. >or ones, particularly glucocorticoids and thyroid hor one, sti ulate proteaso e& ediated protein degradation; insulin opposes these actions. Additionally, cyto'ines, such as tu or necrosis factor *$4)., are capable of increasing uscle proteolysis by #ay of this echanis . In any situations, atrophy is also acco panied by ar'ed increases in the nu ber of autophagic vacuoles. $hese are e brane&bound vacuoles #ithin the cell that contain frag ents of cell co ponents *e.g., itochondria, endoplas ic reticulu . that are destined for destruction and into

#hich the lysoso es discharge their hydrolytic contents. $he cellular co ponents are then digested. 9o e of the cell debris #ithin the autophagic vacuole ay resist digestion and persist as e brane&bound residual bodies that ay re ain as a sarcophagus in the cytoplas . An e"a ple of such residual #odies is the lipofuscin granules, discussed later in the chapter. 7hen present in sufficient a ounts, they i part a bro#n discoloration to the tissue (#rown atrophy). *ETAPLASIA Metaplasia is a reversible change in #hich one adult cell type *epithelial or esenchy al. is replaced by another adult cell type. 1< It ay represent an adaptive substitution of cells that are sensitive to stress by cell types better able to #ithstand the adverse environ ent. $he ost co on epithelial etaplasia is columnar to s%uamous *)ig. 1&CA., as occurs in the respiratory tract in response to chronic irritation. In the habitual cigarette s o'er, the nor al ciliated colu nar epithelial cells of the trachea and bronchi are often replaced focally or #idely by stratified s!ua ous epithelial cells. 9tones in the e"cretory ducts of the salivary glands, pancreas, or bile ducts ay cause replace ent of the nor al secretory colu nar epitheliu by nonfunctioning stratified s!ua ous epitheliu . A deficiency of vita in A *retinoic acid. induces s!ua ous etaplasia in the respiratory epitheliu , and vita in A e"cess suppresses 'eratini%ation *Chapter I.. In all these instances, the ore rugged stratified s!ua ous epitheliu is able to survive under circu stances in #hich the ore fragile speciali%ed colu nar epitheliu ost li'ely #ould have succu bed. Although the etaplastic s!ua ous cells in the respiratory tract, for e"a ple, are capable of surviving, an i portant protective echanis & ucus secretion&is lost. $hus, epithelial etaplasia is a t#o&edged s#ord and, in ost circu stances, represents an undesirable change. Moreover, the influences that predispose to metaplasia, if persistent, may induce malignant transformation in metaplastic epithelium . $hus, the co on for of cancer in the respiratory tract is co posed of s!ua ous cells, #hich arise in areas of etaplasia of the nor al colu nar epitheliu into s!ua ous epitheliu . 'etaplasia from s%uamous to columnar type ay also occur, as in arrett esophagus, in #hich the esophageal s!ua ous epitheliu is replaced by intestinal&li'e colu nar cells under the influence of reflu"ed gastric acid *)ig. 1&C .. Cancers ay arise in these areas, and these are typically glandular *adeno.carcino as *Chapter 1A..

)igure 1&C Metaplasia. A, 9che atic diagra of colu nar to s!ua ous etaplasia. , Metaplastic transfor ation of esophageal stratified s!ua ous epitheliu *left. to ature colu nar epitheliu *so&called Barrett etaplasia..

(onnective tissue metaplasia is the for ation of cartilage, bone, or adipose tissue * esenchy al tissues. in tissues that nor ally do not contain these ele ents. )or e"a ple, bone for ation in uscle, designated myositis ossificans, occasionally occurs after bone fracture. $his type of

etaplasia is less clearly seen as an adaptive response. Mechanisms of Metaplasia. Metaplasia does not result fro a change in the phenotype of a differentiated cell type; instead it is the result of a reprogra ing of ste cells that are 'no#n to e"ist in nor al tissues, or of undifferentiated esenchy al cells present in connective tissue. In a etaplastic change, these precursor cells differentiate along a ne# path#ay. $he differentiation of ste cells to a particular lineage is brought about by signals generated by cyto'ines, gro#th factors, and e"tracellular atri" co ponents in the cell/s environ ent. $issue&specific and differentiation genes are involved in the process, and an increasing nu ber of these are being identified.1@ )or e"a ple, bone orphogenetic proteins, e bers of the $1)&E superfa ily, induce chondrogenic or osteogenic e"pression in ste cells #hile suppressing differentiation into uscle or fat.1, $hese gro#th factors, acting as e"ternal triggers, then induce specific transcription factors that lead the cascade of phenotype&specific genes to#ard a fully differentiated cell. >o# these nor al path#ays run a o' to cause etaplasia is unclear in ost instances. In the case of vita in A deficiency or e"cess, it is 'no#n that retinoic acid regulates cell gro#th, differentiation, and tissue patterning and ay thus influence the differentiation path#ay of ste cells. 1C Certain cytostatic drugs cause a disruption of D4A ethylation patterns and can transfor esenchy al cells fro one type *fibroblast. to another * uscle, cartilage..

/(er(ie! of Cell In%ury and Cell Death

As stated at the beginning of the chapter, cell injury results #hen cells are stressed so severely that they are no longer able to adapt or #hen cells are e"posed to inherently da aging agents. Injury ay progress through a reversible stage and cul inate in cell death *)ig. 1&A.. An overvie# of the orphologic changes in cell injury is sho#n in )igure 1&D. $he bioche ical alterations and the associated orphologic abnor alities are described later, under (Mechanis s of Cell Injury.( $hese alterations ay be divided into the follo#ing stages0 Re$ersi&le cell i#+ ry" Initially, injury is anifested as functional and orphologic changes that are reversible if the da aging sti ulus is re oved. $he hall ar's of reversible injury are reduced o"idative phosphorylation, adenosine triphosphate *A$P. depletion, and cellular s#elling caused by changes in ion concentrations and #ater influ". Irre$ersi&le i#+ ry a#d cell death. 7ith continuing da age, the injury beco es irreversible, at #hich ti e the cell cannot recover. Is there a critical bioche ical event *the (lethal hit(. responsible for the point of no returnJ $here are no clear ans#ers to this !uestion. >o#ever, as discussed later, in ische ic tissues such as the yocardiu , certain structural changes *e.g., a orphous densities in itochondria, indicative of severe itochondrial da age. and functional changes *e.g., loss of e brane per eability. are indicative of cells that have suffered irreversible injury. )rreversi#ly injured cells invaria#ly undergo morphologic changes that are recognized as cell death. $here are t#o types of cell death, necrosis and apoptosis, #hich differ in their orphology, echanis s, and roles in disease and physiology *)ig. 1&I and $able 1&+.. 7hen da age to e branes is severe, lysoso al en%y es enter the cytoplas and digest the cell, and cellular contents lea' out, resulting in necrosis. 9o e no"ious sti uli, especially those that da age D4A, induce another type of death, apoptosis, #hich is characteri%ed by nuclear dissolution #ithout co plete loss of e brane integrity. *hereas necrosis is always a pathologic process, apoptosis serves many normal functions and is not necessarily associated with cell injury . Although #e e phasi%e the distinctions bet#een necrosis and apoptosis, there ay be so e overlaps and co on echanis s bet#een these t#o path#ays. In addition, at least so e types of sti uli ay induce either apoptosis or necrosis, depending on the intensity and duration of the sti ulus, the rapidity of the death process, and the bioche ical derange ents induced in the injured cell. $he echanis s and significance of these t#o death path#ays are discussed later in the chapter.

)igure 1&A 9tages in the evolution of cell injury and death.

In the follo#ing sections, #e #ill discuss the causes and echanis s of cell injury. 7e first describe the se!uence of events in cell injury and its co on end point, necrosis, and discuss selected illustrative e"a ples of cell injury and necrosis. 7e conclude #ith a discussion of the uni!ue pattern of cell death represented by apoptosis.

Causes of Cell In%ury

$he causes of cell injury range fro the e"ternal gross physical violence of an auto obile accident to internal endogenous causes, such as a subtle genetic utation causing lac' of a vital en%y e that i pairs nor al etabolic function. Most injurious sti uli can be grouped into the follo#ing broad categories. /*ygen Depri(ation. Hypoxia is a deficiency of o"ygen, #hich causes cell injury by reducing aerobic o"idative respiration. >ypo"ia is an e"tre ely i portant and co on cause of cell injury and cell death. It should be distinguished fro ischemia, #hich is a loss of blood supply fro i peded arterial flo# or reduced venous drainage in a tissue. Ische ia co pro ises the supply not only of o"ygen, but also of etabolic substrates, including glucose *nor ally provided by flo#ing blood.. $herefore, ische ic tissues are injured ore rapidly and severely than are hypo"ic tissues. :ne cause of hypo"ia is inade!uate o"ygenation of the blood due to cardiorespiratory failure. Koss of the o"ygen&carrying capacity of the blood, as in ane ia or carbon ono"ide poisoning *producing a stable carbon ono"yhe oglobin that bloc's o"ygen carriage., is a less fre!uent cause of o"ygen deprivation that results in significant injury. Depending on the severity of the hypo"ic state, cells ay adapt, undergo injury, or die. )or e"a ple, if the fe oral artery is narro#ed, the s'eletal uscle cells of the leg ay shrin' in si%e *atrophy.. $his reduction in cell ass achieves a balance bet#een etabolic needs and the available o"ygen supply. More severe hypo"ia induces injury and cell death.
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)igure 1&D 9che atic representation of a nor al cell and the changes in reversible and irreversible cell injury. Depicted are orphologic changes, #hich are described in the follo#ing pages and sho#n in electron icrographs in )igure 1&1A. 6eversible injury is characteri%ed by generali%ed s#elling of the cell and its organelles; blebbing of the plas a e brane; detach ent of riboso es fro the endoplas ic reticulu ; and clu ping of nuclear chro atin. $ransition to irreversible injury is characteri%ed by increasing s#elling of the cell; s#elling and disruption of lysoso es; presence of large a orphous densities in s#ollen itochondria; disruption of cellular e branes; and profound nuclear changes. $he latter include nuclear codensation *py'nosis., follo#ed by frag entation *'aryorrhe"is. and dissolution of the nucleus *'aryolysis.. Ka inated structures * yelin figures. derived fro da aged e branes of organelles and the plas a e brane first appear during the reversible stage and beco e ore pronounced in irreversibly da aged cells. $he echanis s underlying these changes are discussed in the te"t that follo#s.

Physical gents. Physical agents capable of causing cell injury include echanical trau a, e"tre es of te perature *burns and deep cold., sudden changes in at ospheric pressure, radiation, and electric shoc' *Chapter I..
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)igure 1&I $he se!uential ultrastructural changes seen in necrosis (left) and apoptosis (right). In apoptosis, the initial changes consist of nuclear chro atin condensation and frag entation, follo#ed by cytoplas ic budding and phagocytosis of the e"truded apoptotic bodies. 9igns of cytoplas ic blebs, and digestion and lea'age of cellular co ponents. *Adapted fro 7al'er 4I, et al0 Patterns of cell death. Methods Archiv 3"p Pathol 1<01D&<+, 1IDD. 6eproduced #ith per ission of 9. 2arger A1, Basel..

Ta&le "#0. Features of Necrosis and poptosis

Feature Cell si%e 4ucleus

Necrosis 3nlarged *s#elling.

poptosis 6educed *shrin'age.

Py'nosis ? 'aryorrhe"is )rag entation into nucleoso e si%e ? 'aryolysis frag ents Disrupted Intact; altered structure, especially orientation of lipids Intact; ay be released in apoptotic bodies

Plas a e brane

Cellular contents 3n%y atic digestion; ay lea' out of cell Adjacent infla ation Physiologic or pathologic role )re!uent

4o

Invariably pathologic *cul ination of irreversible cell injury.

:ften physiologic, eans of eli inating un#anted cells; ay be pathologic after so e for s of cell injury, especially D4A da age

Chemical gents and Drugs. $he list of che icals that ay produce cell injury defies co pilation. 9i ple che icals such as glucose or salt in hypertonic concentrations ay cause cell injury directly or by deranging electrolyte ho eostasis of cells. 3ven o"ygen, in high concentrations, is severely to"ic. $race a ounts of agents 'no#n as poisons, such as arsenic, cyanide, or ercuric salts, ay destroy sufficient nu bers of cells #ithin inutes to hours to cause death. :ther substances, ho#ever, are our daily co panions0 environ ental and air pollutants, insecticides, and herbicides; industrial and occupational ha%ards, such as carbon ono"ide and asbestos; social sti uli, such as alcohol and narcotic drugs; and the ever&increasing variety of therapeutic drugs. Infectious gents. $hese agents range fro the sub icroscopic viruses to the large tape#or s. In bet#een are the ric'ettsiae, bacteria, fungi, and higher for s of parasites. $he #ays by #hich this heterogeneous group of biologic agents cause injury are diverse and are discussed in Chapter D. Immunologic 'eactions. Although the i une syste serves an essential function in defense against infectious pathogens, i une reactions ay, in fact, cause cell injury. $he anaphylactic reaction to a foreign protein or a drug is a pri e e"a ple, and reactions to endogenous self& antigens are responsible for a nu ber of autoi une diseases *Chapter C.. Genetic Derangements. 1enetic defects as causes of cell injury are of ajor interest to scientists and physicians today *Chapter ,.. $he genetic injury ay result in a defect as severe as the congenital alfor ations associated #ith Do#n syndro e, caused by a chro oso al abnor ality, or as subtle as the decreased life of red blood cells caused by a single a ino acid substitution in he oglobin 9 in sic'le cell ane ia. $he any inborn errors of etabolis arising fro en%y atic abnor alities, usually an en%y e lac', are e"cellent e"a ples of cell da age due to subtle alterations at the level of D4A. 5ariations in the genetic a'eup can also influence the susceptibility of cells to injury by che icals and other environ ental insults.
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)igure 1&1- Cellular and bioche ical sites of da age in cell injury.

Nutritional Im&alances. 4utritional i balances continue to be ajor causes of cell injury. Protein& calorie deficiencies cause an appalling nu ber of deaths, chiefly a ong underprivileged populations. Deficiencies of specific vita ins are found throughout the #orld *Chapter I.. 4utritional proble s can be self&i posed, as in anore"ia nervosa or self&induced starvation. Ironically, nutritional e"cesses have also beco e i portant causes of cell injury. 3"cesses of lipids predispose to atherosclerosis, and obesity is a anifestation of the overloading of so e cells in the body #ith fats. Atherosclerosis is virtually ende ic in the Bnited 9tates, and obesity is ra pant. In addition to the proble s of undernutrition and overnutrition, the co position of the diet a'es a significant contribution to a nu ber of diseases. Metabolic diseases such as diabetes also cause severe cell injury.

Mechanisms of Cell In%ury

$he bioche ical echanis s responsible for cell injury are co ple". $here are, ho#ever, a nu ber of principles that are relevant to ost for s of cell injury0 +he cellular response to injurious stimuli depends on the type of injury, its duration, and its severity. $hus, s all doses of a che ical to"in or brief periods of ische ia ay induce reversible injury, #hereas large doses of the sa e to"in or ore prolonged ische ia ight result either in instantaneous cell death or in slo#, irreversible injury leading in ti e to cell death. +he conse%uences of cell injury depend on the type, state, and adapta#ility of the injured cell. $he cell/s nutritional and hor onal status and its etabolic needs are i portant in its response to injury. >o# vulnerable is a cell, for e"a ple, to loss of blood supply and hypo"iaJ $he striated uscle cell in the leg can be placed entirely at rest #hen it is deprived of its blood supply; not so the striated uscle of the heart. 3"posure of t#o individuals to identical concentrations of a to"in, such as carbon tetrachloride, ay produce no effect in one and cell death in the other. $his ay be due to genetic variations affecting the a ount and activity of hepatic en%y es that convert carbon tetrachloride to to"ic byproducts *Chapter I.. 7ith the co plete apping of the hu an geno e, there is great interest in identifying genetic poly orphis s that affect the cell/s response to injurious agents. (ell injury results from functional and #iochemical a#normalities in one or more of several essential cellular components *)ig. 1&1-.. $he ost i portant targets of injurious sti uli are0 *1. aerobic respiration involving itochondrial o"idative phosphorylation and production of A$P; *+. the integrity of cell e branes, on #hich the ionic and os otic ho eostasis of the cell and its organelles depends; *<. protein synthesis; *@. the cytos'eleton; and *,. the integrity of the genetic apparatus of the cell.

In the follo#ing section, #e describe each of the bioche ical echanis s that are responsible for cell injury induced by different sti uli. It should be noted that #ith ost sti uli, ultiple echanis s contribute to injury, and in the case of any injurious sti uli, the actual bioche ical locus of injury re ains un'no#n. DEPLETIO, O- ATP
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)igure 1&11 )unctional and

orphologic conse!uences of decreased intracellular A$P during cell injury.

A+P depletion and decreased A+P synthesis are fre%uently associated with #oth hypoxic and chemical (toxic) injury *)ig. 1&11.. >igh&energy phosphate in the for of A$P is re!uired for any synthetic and degradative processes #ithin the cell. $hese include e brane transport, protein

synthesis, lipogenesis, and the deacylation&reacylation reactions necessary for phospholipid turnover. A$P is produced in t#o #ays. $he ajor path#ay in a alian cells is oxidative phosphorylation of adenosine diphosphate, in a reaction that results in reduction of o"ygen by the electron transfer syste of itochondria. $he second is the glycolytic pathway, #hich can generate A$P in the absence of o"ygen using glucose derived either fro body fluids or fro the hydrolysis of glycogen. $hus, tissues #ith greater glycolytic capacity *e.g., liver. have an advantage #hen A$P levels are falling because of inhibition of o"idative etabolis by injury. Depletion of A$P to L,M to 1-M of nor al levels has #idespread effects on any critical cellular syste s0 $he activity of the plasma mem#rane energy&dependent sodium pump *ouabain&sensitive 4aN, 2N&A$Pase. is reduced. )ailure of this active transport syste , due to di inished A$P concentration and enhanced A$Pase activity, causes sodiu to accu ulate intracellularly and potassiu to diffuse out of the cell. $he net gain of solute is acco panied by isos otic gain of #ater, causing cell swelling, and dilation of the endoplas ic reticulu *see )ig. 1&D.. (ellular energy meta#olism is altered . If the supply of o"ygen to cells is reduced, as in ische ia, o"idative phosphorylation ceases and cells rely on glycolysis for energy production. $his s#itch to anaerobic etabolis is controlled by energy path#ay etabolites acting on glycolytic en%y es. $he decrease in cellular A$P and associated increase in adenosine onophosphate sti ulate phosphofructo'inase and phosphorylase activities. $hese result in an increased rate of anaero#ic glycolysis designed to aintain the cell/s energy sources by generating A$P through etabolis of glucose derived fro glycogen. As a conse!uence, glycogen stores are rapidly depleted . 1lycolysis results in the accu ulation of lactic acid and inorganic phosphates fro the hydrolysis of phosphate esters. $his reduces the intracellular p>, resulting in decreased activity of any cellular en%y es. )ailure of the Ca+N pu p leads to influ" of Ca +N, #ith da aging effects on nu erous cellular co ponents, described belo#. 7ith prolonged or #orsening depletion of A$P, structural disruption of the protein synthetic apparatus occurs, anifested as detach ent of riboso es fro the rough endoplas ic reticulu and dissociation of polyso es into onoso es, #ith a conse!uent reduction in protein synthesis. Blti ately, there is irreversible da age to itochondrial and lysoso al e branes, and the cell undergoes necrosis *see )ig. 1&D.. In cells deprived of o"ygen or glucose , proteins ay beco e isfolded, and isfolded proteins trigger a cellular reaction called the unfolded protein response that ay lead to cell injury and even death. $his process is described later in the chapter. Protein isfolding is also seen in cells e"posed to stress, such as heat, and #hen proteins are da aged by en%y es *such as Ca+N&responsive en%y es, described belo#. and free radicals.

*ITO.HO,DRIAL DA*A/E Mitochondria are i portant targets for virtually all types of injurious sti uli, including hypo"ia and to"ins. Cell injury is fre!uently acco panied by orphologic changes in itochondria. Mitochondria can be da aged by increases of cytosolic Ca +N, by o"idative stress, by brea'do#n of phospholipids through the phospholipase A + and sphingo yelin path#ays, and by lipid brea'do#n products derived therefro , such as free fatty acids and cera ide. Mitochondrial da age often results in the for ation of a high&conductance channel, the so&called itochondrial per eability transition, in the inner itochondrial e brane *)ig. 1&1+..1A Although reversible in its early stages, this nonselective pore beco es per anent if the inciting sti uli persist, precluding aintenance of itochondrial proton otive force, or potential. Because aintenance of e brane potential is critical for itochondrial o"idative phosphorylation, it follo#s that irreversible itochondrial per eability transition is a deathblo# to the cell. Mitochondrial da age can also be

associated #ith lea'age of cytochro e c into the cytosol. Because cytochro e c is an integral co ponent of the electron transport chain and can trigger apoptotic death path#ays in the cytosol *see later., this pathologic event is also li'ely to be a 'ey deter inant of cell death. I,-L01 O- I,TRA.ELL0LAR .AL.I0* A,D LOSS O- .AL.I0* HO*EOSTASIS
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)igure 1&1+ Mitochondrial dysfunction in cell injury. )igure 1&1< 9ources and conse!uences of increased cytosolic calciu adenosine triphosphate. in cell injury. A$P,

Calciu ions are i portant ediators of cell injury. 1D Cytosolic free calciu is aintained at e"tre ely lo# concentrations *L-.1 O ol. co pared #ith e"tracellular levels of 1.< ol, and ost intracellular calciu is se!uestered in itochondria and endoplas ic reticulu . 9uch gradients are odulated by e brane&associated, energy&dependent Ca +N, Mg+N&A$Pases. Ische ia and certain to"ins cause an early increase in cytosolic calciu concentration, o#ing to the net influ" of Ca +N across the plas a e brane and the release of Ca +N fro itochondria and endoplas ic reticulu *)ig. 1&1<.. 9ustained rises in intracellular Ca+N subse!uently result fro nonspecific increases in e brane per eability. Increased Ca +N in turn activates a nu ber of en%y es, #ith potential deleterious cellular effects. $he en%y es 'no#n to be activated by calciu include A+Pases *thereby hastening A$P depletion., phospholipases *#hich cause e brane da age., proteases *#hich brea' do#n both e brane and cytos'eletal proteins., and endonucleases *#hich are responsible for D4A and chro atin frag entation.. Increased intracellular Ca+N levels also result in increased itochondrial per eability and the induction of apoptosis.1D&+- Although cell injury often results in increased intracellular calciu and this in turn ediates a variety of deleterious effects, including cell death, loss of calciu ho eostasis is not al#ays a pro"i al event in irreversible cell injury. A..0*0LATIO, O- O1Y/E,2DERI3ED -REE RADI.ALS (O1IDATI3E STRESS! Cells generate energy by reducing olecular o"ygen to #ater. During this process, s all a ounts of partially reduced reactive o"ygen for s are produced as an unavoidable byproduct of itochondrial respiration. 9o e of these for s are free radicals that can da age lipids, proteins, and nucleic acids. $hey are referred to as reactive oxygen species. Cells have defense syste s to prevent injury caused by these products. An i balance bet#een free radical&generating and radical&scavenging syste s results in oxidative stress, a condition that has been associated #ith the cell injury seen in any pathologic conditions. )ree radical& ediated da age contributes to such varied processes as che ical and radiation injury, ische ia&reperfusion injury *induced by restoration of blood flo# in ische ic tissue., cellular aging, and icrobial 'illing by phagocytes. +1&+@ )ree radicals are che ical species that have a single unpaired electron in an outer orbit. 3nergy created by this unstable configuration is released through reactions #ith adjacent olecules, such as inorganic or organic che icals&proteins, lipids, carbohydrates&particularly #ith 'ey olecules in e branes and nucleic acids. Moreover, free radicals initiate autocatalytic reactions, #hereby olecules #ith #hich they react are the selves converted into free radicals to propagate the chain of da age. Free radicals may &e i#itiated !ithin cells in se(eral !ays *)ig. 1&1@.0 A&sorptio# of radia#t e#ergy *e.g., ultraviolet light, "&rays.. )or e"a ple, ioni%ing radiation can hydroly%e #ater into hydro"yl *:>. and hydrogen *>. free radicals. E#4y%atic %eta&olis% of e5oge#o s che%icals or dr gs *e.g., carbon tetrachloride FCCl@G can generate CCl<, described later in this chapter.. The red ctio#2o5idatio# reactio#s that occ r d ri#g #or%al %eta&olic processes . During nor al respiration, olecular o"ygen is se!uentially reduced by the addition of four electrons to generate #ater. 9uch conversion occurs by o"idative en%y es in the

endoplas ic reticulu , cytosol, itochondria, pero"iso es, and lysoso es. In this process, s all a ounts of to"ic inter ediates are produced; these include supero"ide anion radical *:+&., hydrogen pero"ide *>+:+., and hydro"yl ions *:>.. 6apid bursts of supero"ide production occur in activated poly orphonuclear leu'ocytes during infla ation. $his occurs by a precisely controlled reaction in a plas a e brane ultiprotein co ple" that uses 4ADP> o"idase for the redo" reaction *Chapter +.. In addition, so e intracellular o"idases *such as "anthine o"idase. generate supero"ide radicals as a conse!uence of their activity. Tra#sitio# %etals such as iron and copper donate or accept free electrons during intracellular reactions and cataly%e free radical for ation, as in the )enton reaction *> +:+ N )e+N ? )e<N N :> N :>&.. Because ost of the intracellular free iron is in the ferric *)e <N. state, it ust be first reduced to the ferrous *)e +N. for to participate in the )enton reaction. $his reduction can be enhanced by supero"ide, and thus sources of iron and supero"ide are re!uired for a"i al o"idative cell da age. ,itric o5ide 1N/2, an i portant che ical ediator generated by endothelial cells, acrophages, neurons, and other cell types *Chapter +., can act as a free radical and can also be converted to highly reactive pero"ynitrite anion *:4:: &. as #ell as 4:+ and 4:<&.
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$he effects of these reactive species are #ide&ranging, but three reactions are particularly relevant to cell injury *see )ig. 1&1@.0 Lipid pero5idatio# of %e%&ra#es. )ree radicals in the presence of o"ygen ay cause pero"idation of lipids #ithin plas a and organellar e branes. :"idative da age is initiated #hen the double bonds in unsaturated fatty acids of e brane lipids are attac'ed by o"ygen&derived free radicals, particularly by :>. $he lipid&free radical interactions yield pero"ides, #hich are the selves unstable and reactive, and an autocatalytic chain reaction ensues *called propagation., #hich can result in e"tensive e brane, organellar, and cellular da age. :ther ore favorable ter ination options ta'e place #hen the free radical is captured by a scavenger, such as vita in 3 , e bedded in the cell e brane. O5idati$e %odificatio# of protei#s. )ree radicals pro ote o"idation of a ino acid residue side chains, for ation of protein&protein cross&lin'ages *e.g., disulfide bonds., and o"idation of the protein bac'bone, resulting in protein frag entation. :"idative odification enhances degradation of critical proteins by the ulticatalytic proteaso e co ple", raising havoc throughout the cell. Lesio#s i# D,A. 6eactions #ith thy ine in nuclear and itochondrial D4A produce single&stranded brea's in D4A. $his D4A da age has been i plicated in cell aging *discussed later in this chapter. and in alignant transfor ation of cells *Chapter A..

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)igure 1&1@ $he role of reactive o"ygen species in cell injury. :+ is converted to supero"ide *:+&. by o"idative en%y es in the endoplas ic reticulu *36., itochondria, plas a e brane, pero"iso es, and cytosol. :+& is converted to >+:+ by dis utation and thence to :> by the Cu+NP)e+N&cataly%ed )enton reaction. >+:+ is also derived directly fro o"idases in pero"iso es. 4ot sho#n is another potentially injurious radical, singlet o"ygen. 6esultant free radical da age to lipid *pero"idation., proteins, and D4A leads to various for s of cell injury. 4ote that supero"ide cataly%es the reduction of )e<N to )e+N, thus enhancing :> generation by the )enton reaction. $he ajor antio"idant en%y es are supero"ide dis utase *9:D., catalase, and glutathione pero"idase. 19>, reduced glutathione; 1991, o"idi%ed glutathione; 4ADP>, reduced for of nicotina ide adenine dinucleotide phosphate.

.ells ha$e de$eloped % ltiple %echa#is%s to re%o$e free radicals a#d there&y %i#i%i4e i#+ ry. )ree radicals are inherently unstable and generally decay spontaneously. 9upero"ide, for e"a ple, is unstable and decays *dis utates. spontaneously into o"ygen and hydrogen pero"ide in the presence of #ater. $here are, ho#ever, several nonen%y atic and en%y atic syste s that contribute to inactivation of free radical reactions *see )ig. 1&1@.. $hese include the follo#ing0 A#tio5ida#ts either bloc' the initiation of free radical for ation or inactivate *e.g., scavenge. free radicals and ter inate radical da age. 3"a ples are the lipid&soluble vita ins 3 and A as #ell as ascorbic acid and glutathione in the cytosol. As #e have seen, iro# and copper can cataly%e the for ation of reactive o"ygen species. $he levels of these reactive for s are ini i%ed by binding of the ions to storage and transport proteins *e.g., transferrin, ferritin, lactoferrin, and ceruloplas in., thereby ini i%ing :> for ation. A series of e#4y%es acts as free radical&scavenging syste s and brea' do#n hydrogen pero"ide and supero"ide anion. $hese en%y es are located near the sites of generation of these o"idants and include the follo#ing0 o .atalase, present in pero"iso es, #hich deco poses > +:+ *+ >+:+ ? :+ N + >+:.. o S pero5ide dis% tases are found in any cell types and convert supero"ide to >+:+ *+ :+& N + > ? >+:+ N :+..+< $his group includes both anganese& supero"ide dis utase, #hich is locali%ed in itochondria, and copper&%inc& supero"ide dis utase, #hich is found in the cytosol. o /l tathio#e pero5idase also protects against injury by cataly%ing free radical brea'do#n *>+:+ N + 19> ? 1991 Fglutathione ho odi erG N + > +:, or + :> N + 19> ? 1991 N + >+:.. $he intracellular ratio of o"idi%ed glutathione *1991. to reduced glutathione *19>. is a reflection of the o"idative state of the cell and is an i portant aspect of the cell/s ability to deto"ify reactive o"ygen species.

In any pathologic processes, the final effects induced by free radicals depend on the net balance bet#een free radical for ation and ter ination. As stated earlier, free radicals are thought to be involved in any pathologic and physiologic processes, to be entioned throughout this boo'. DE-E.TS I, *E*(RA,E PER*EA(ILITY

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)igure 1&1, Mechanis s of e brane da age in cell injury. Decreased : + and increased cytosolic Ca+N are typically seen in ische ia but ay acco pany other for s of cell injury. 6eactive o"ygen species, #hich are often produced on reperfusion of ische ic tissues, also cause e brane da age *not sho#n..

3arly loss of selective e brane per eability leading ulti ately to overt e brane da age is a consistent feature of ost for s of cell injury. Me brane da age ay affect the itochondria, the plas a e brane, and other cellular e branes. In ische ic cells, e brane defects ay be the result of a series of events involving A$P depletion and calciu & odulated activation of phospholipases *see belo#.. $he plas a e brane, ho#ever, can also be da aged directly by certain bacterial to"ins, viral proteins, lytic co ple ent co ponents, and a variety of physical and che ical agents. 9everal bioche ical echanis s ay contribute to e brane da age *)ig. 1& 1,.0 *itocho#drial dysf #ctio#. Defective itochondrial function results in decreased phospholipid synthesis, #hich affects all cellular e branes, including the itochondria the selves. At the sa e ti e, increase of cytosolic calciu associated #ith A$P depletion results in increased upta'e of Ca +N into the itochondria, activating phospholipases and leading to brea'do#n of phospholipids. $he net result is depletion of phospholipids fro

the itochondria and other cellular e branes, and accu ulation of free fatty acids. In the itochondria, these changes cause per eability defects, such as the itochondrial per eability transition,1A,+C leading to progresive cell injury *see )ig. 1&1+.. Loss of %e%&ra#e phospholipids. 9evere cell injury is associated #ith a decrease in the content of e brane phospholipids, because of degradation li'ely due to activation of endogenous phospholipases by increased levels of cytosolic calciu . +C Phospholipid loss can also occur secondary to decreased A$P&dependent reacylation or di inished de novo synthesis of phospholipids. .ytoskeletal a&#or%alities. Cytos'eletal fila ents serve as anchors connecting the plas a e brane to the cell interior. Activation of proteases by increased cytosolic calciu ay cause da age to ele ents of the cytos'eleton. In the presence of cell s#elling, this da age results, particularly in yocardial cells, in detach ent of the cell e brane fro the cytos'eleton, rendering it susceptible to stretching and rupture. Reacti$e o5yge# species. Partially reduced o"ygen free radicals cause injury to cell e branes and other cell constituents, by echanis s that #ere discussed earlier. Lipid &reakdow# prod cts. $hese include unesterified free fatty acids, acyl carnitine, and lysophospholipids, catabolic products that are 'no#n to accu ulate in injured cells as a result of phospholipid degradation. $hey have a detergent effect on e branes. $hey also either insert into the lipid bilayer of the e brane or e"change #ith e brane phospholipids, potentially causing changes in per eability and electrophysiologic alterations.1A
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Da age to itochondrial e branes has conse!uences that #ere described above. Plas a e brane da age results in loss of os otic balance and influ" of fluids and ions, as #ell as loss of proteins, en%y es, coen%y es, and ribonucleic acids. $he cells ay also lea' etabolites, #hich are vital for the reconstitution of A$P, thus further depleting net intracellular high&energy phosphates. )njury to lysosomal mem#ranes results in lea"age of their enzymes into the cytoplasm and activation of these enzymes. Kysoso es contain 64ases, D4ases, proteases, phosphatases, glucosidases, and cathepsins. Activation of these en%y es leads to en%y atic digestion of cell co ponents, resulting in loss of ribonucleoprotein, deo"yribonucleoprotein, and glycogen, and the cells die by necrosis.

'e(ersi&le and Irre(ersi&le Cell In%ury

Bp to this point, #e have focused on the causes and general bioche ical echanis s of cell injury. In this section, #e #ill turn our attention to the path#ays underlying the se!uence of events #hereby reversi#le injury #ecomes irreversi#le, leading to cell death, principally necrosis. As discussed previously, the earliest changes associated #ith various for s of cell injury are decreased generation of A$P, loss of cell e brane integrity, defects in protein synthesis, cytos'eletal da age, and D4A da age. 7ithin li its, the cell can co pensate for these derange ents and, if the injurious sti ulus abates, #ill return to nor alcy. Persistent or e"cessive injury, ho#ever, causes cells to pass the threshold into irreversi#le injury *see )ig. 1&D.. $his is associated #ith e"tensive da age to all cellular e branes, s#elling of lysoso es, and vacuoli%ation of itochondria #ith reduced capacity to generate A$P. 3"tracellular calciu enters the cell and intracellular calciu stores are released, resulting in the activation of en%y es that can cataboli%e e branes, proteins, A$P, and nucleic acids. )ollo#ing this, there is continued loss of proteins, essential coen%y es, and ribonucleic acids fro the hyperper eable plas a e brane, #ith cells lea'ing etabolites vital for the reconstitution of A$P and further depleting intracellular high&energy phosphates. $he olecular echanis s connecting ost for s of cell injury to ulti ate cell death have proved

elusive, for several reasons. )irst, there are clearly any #ays to injure a cell, not all of the invariably fatal. 9econd, the nu erous acro olecules, en%y es, and organelles #ithin the cell are so closely interdependent that it is difficult to distinguish a pri ary injury fro secondary *and not necessarily relevant. ripple effects. $hird, the (point of no return,( at #hich irreversible da age has occurred, is still largely undeter ined; thus, #e have no precise cut&off point to establish cause and effect. )inally, there is probably no single co on final path#ay by #hich cells die. It is, therefore, difficult to define the stage beyond #hich the cell is irretrievably doo ed to destruction. And #hen does the cell actually dieJ $#o pheno ena consistently characteri%e irreversibility. +he first is the ina#ility to reverse mitochondrial dysfunction *lac' of o"idative phosphorylation and A$P generation. even after resolution of the original injury. +he second is the development of profound distur#ances in mem#rane function. As entioned earlier, injury to the lysoso al e branes results in lea'age of their en%y es into the cytoplas ; the acid hydrolases are activated in the reduced intracellular p> of the ische ic cell and degrade cytoplas ic and nuclear co ponents. $his dissolution of the injured cell is characteristic of necrosis, one of the recogni%ed patterns of cell death. $here is also #idespread lea'age of potentially destructive cellular en%y es into the e"tracellular space, #ith da age to adjacent tissues and a host response *Chapter +.. 7hatever the echanis *s. of e brane da age, the end result is a assive lea' of intracellular aterials and a assive influ" of calciu , #ith the conse!uences described above. It is #orth noting that lea'age of intracellular proteins across the degraded cell e brane into the peripheral circulation provides a eans of detecting tissue&specific cellular injury and death using blood seru sa ples. Cardiac uscle, for e"a ple, contains a specific isofor of the en%y e creatine 'inase and of the contractile protein troponin; liver *and specifically bile duct epitheliu . contains a te perature&resistant isofor of the en%y e al'aline phosphatase; and hepatocytes contain transa inases. Irreversible injury and cell death in these tissues are conse!uently reflected in increased levels of such proteins in the blood.

Morphology of Cell In%ury and Necrosis

Cells undergo se!uential bioche ical and orphologic changes as they are progressively injured and ulti ately die by necrosis. All stresses and no"ious influences e"ert their effects first at the olecular or bioche ical level. $here is a ti e lag bet#een the stress and the orphologic changes of cell injury or death; the duration of this delay ay vary #ith the sensitivity of the ethods used to detect these changes *)ig. 1&1C.. 7ith histoche ical or ultrastructural techni!ues, changes ay be seen in inutes to hours after ische ic injury; ho#ever, it ay ta'e considerably longer *hours to days. before changes can be seen by light icroscopy or on gross e"a ination. As #ould be e"pected, the orphologic anifestations of necrosis ta'e ore ti e to develop than those of reversible da age. )or e"a ple, cell s#elling is a reversible orphologic change, and this ay occur in a atter of inutes. Bn ista'able light icroscopic changes of cell death, ho#ever, do not occur in the yocardiu until @ to 1+ hours after total ische ia, yet #e 'no# that irreversible injury occurs #ithin +- to C- inutes. Re$ersi&le I#+ ry

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)igure 1&1C $i ing of bioche ical and

orphologic changes in cell injury.

$#o patterns of reversible cell injury can be recogni%ed under the light icroscope0 cell lar swelli#g and fatty cha#ge. Cellular s#elling appears #henever cells are incapable of aintaining ionic and fluid ho eostasis and is the result of loss of function of plas a e brane energy& dependent ion pu ps. )atty change occurs in hypo"ic injury and various for s of to"ic or

etabolic injury. It is anifested by the appearance of s all or large lipid vacuoles in the cytoplas and occurs in hypo"ic and various for s of to"ic injury. It is principally encountered in cells involved in and dependent on fat etabolis , such as the hepatocyte and yocardial cell. $he echanis s of fatty change are discussed in ore detail later in the chapter. Morphology. Cellular s#elling is the first anifestation of al ost all for s of injury to cells. It is a difficult orphologic change to appreciate #ith the light icroscope; it ay be ore apparent at the level of the #hole organ. 7hen it affects any cells in an organ, it causes so e pallor, increased turgor, and increase in #eight of the organ. :n icroscopic e"a ination, s all clear vacuoles ay be seen #ithin the cytoplas ; these represent distended and pinched&off seg ents of the endoplas ic reticulu . $his pattern of nonlethal injury is so eti es called hydropic change or vacuolar degeneration. 9#elling of cells is reversible. $he ultrastructural changes of reversible cell injury *)ig. 1&1A. include0 1. plasma mem&rane alterations, such as blebbing, blunting, and distortion of icrovilli; creation of yelin figures; and loosening of intercellular attach ents +. mitochondrial changes, including s#elling, rarefaction, and the appearance of s all phospholipid&rich a orphous densities <. dilation of the endoplasmic reticulum, #ith detach ent and disaggregation of polyso es @. nuclear alterations, #ith disaggregation of granular and fibrillar ele ents.

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)igure 1&1A Morphologic changes in reversible and irreversible cell injury. A, 3lectron icrograph of a nor al epithelial cell of the pro"i al 'idney tubule. 4ote abundant icrovilli * v. lining the lu en *K.. 4, nucleus; 5, apical vacuoles *#hich are nor al structures in this cell type.. , 3pithelial cell of the pro"i al tubule sho#ing reversible ische ic changes. $he icrovilli * v. are lost and have been incorporated in apical cytoplas ; blebs have for ed and are e"truded in the lu en *K.. Mitochondria are slightly dilated. *Co pare #ith A.. (, Pro"i al tubular cell sho#ing irreversible ische ic injury. 4ote the ar'edly s#ollen itochondria containing a orphous densities, disrupted cell e branes, and dense py'notic nucleus. *Courtesy of Dr. M.A. 5en'atachala , Bniversity of $e"as, 9an Antonio, $Q..

,ecrosis 4ecrosis refers to a spectru of orphologic changes that follo# cell death in living tissue, largely resulting fro the progressive degradative action of en%y es on the lethally injured cell *cells placed i ediately in fi"ative are dead but not necrotic.. As co only used, necrosis is the gross and histologic correlate of cell death occurring in the setting of irreversible e"ogenous injury. 4ecrotic cells are unable to aintain e brane integrity and their contents often lea' out. $his ay elicit infla ation in the surrounding tissue. $he orphologic appearance of necrosis is the result of denaturation of intracellular proteins and en%y atic digestion of the cell. $he en%y es are derived either fro the lysoso es of the dead cells the selves, in #hich case the en%y atic digestion is referred to as autolysis, or fro the lysoso es of i igrant leu'ocytes, during infla atory reactions. $hese processes re!uire hours to develop, and so there #ould be no detectable changes in cells if, for e"a ple, a yocardial infarct caused sudden death. $he only telling evidence ight be occlusion of a coronary artery. $he earliest histologic evidence of yocardial necrosis does not beco e anifest until @ to 1+ hours later, but cardiac&specific en%y es and proteins that are released fro necrotic uscle can

be detected in the blood as early as + hours after

yocardial cell death.

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Morphology. 4ecrotic cells sho# increased eosinophilia attributable in part to loss of the nor al basophilia i parted by the 64A in the cytoplas and in part to the increased binding of eosin to denatured intracytoplas ic proteins *)ig. 1&1D.. $he necrotic cell ay have a ore glassy ho ogeneous appearance than that of nor al cells, ainly as a result of the loss of glycogen particles. 7hen en%y es have digested the cytoplas ic organelles, the cytoplas beco es vacuolated and appears oth&eaten. )inally, calcification of the dead cells ay occur. Dead cells ay ulti ately be replaced by large, #horled phospholipid asses called myelin figures. $hese phospholipid precipitates are then either phagocytosed by other cells or further degraded into fatty acids; calcification of such fatty acid residues results in the generation of calciu soaps. By electron icroscopy, necrotic cells are characteri%ed by overt discontinuities in plas a and organelle e branes, ar'ed dilation of itochondria #ith the appearance of large a orphous densities, intracytoplas ic yelin figures, a orphous os iophilic debris, and aggregates of fluffy aterial probably representing denatured protein *see )ig. 1&1A(.. Nuclear changes appear in the for of one of three patterns, all due to nonspecific brea'do#n of D4A *see )igs. 1&D and 1&1A(.. $he basophilia of the chro atin ay fade 13aryolysis2, a change that presu ably reflects D4ase activity. A second pattern *also seen in apoptotic cell death. is py3nosis, characteri%ed by nuclear shrin'age and increased basophilia. >ere the D4A apparently condenses into a solid, shrun'en basophilic ass. In the third pattern, 'no#n as 3aryorrhe*is, the py'notic or partially py'notic nucleus undergoes frag entation. 7ith the passage of ti e *a day or t#o., the nucleus in the necrotic cell totally disappears. :nce the necrotic cells have undergone the early alterations described, the ass of necrotic cells ay have several orphologic patterns. Although the ter s are so e#hat out oded, they are routinely used and their eanings are understood by both pathologists and clinicians. 7hen denaturation is the pri ary pattern, coagulati(e necrosis develops. In the instance of do inant en%y e digestion, the result is li-uefacti(e necrosis+ in special circu stances, caseous necrosis and fat necrosis ay occur.
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Coagulati(e necrosis i plies preservation of the basic outline of the coagulated cell for a span of at least so e days *)ig. 1&1IA.. $he affected tissues e"hibit a fir te"ture. Presu ably, the injury or the subse!uent increasing intracellular acidosis denatures not only structural proteins but also en%y es and so bloc's the proteolysis of the cell. $he yocardial infarct is an e"cellent e"a ple in #hich acidophilic, coagulated, anucleate cells ay persist for #ee's. Blti ately, the necrotic yocardial cells are re oved by frag entation and phagocytosis of the cellular debris by scavenger leu'ocytes and by the action of proteolytic lysoso al en%y es brought in by the i igrant #hite cells. $he process of coagulative necrosis, #ith preservation of the general tissue architecture, is characteristic of hypo"ic death of cells in all tissues e"cept the brain. 4i-uefacti(e necrosis is characteristic of focal bacterial or, occasionally, fungal infections, because icrobes sti ulate the accu ulation of

infla atory cells *)ig. 1&1I .. )or obscure reasons, hypo"ic death of cells #ithin the central nervous syste often evo'es li!uefactive necrosis. 7hatever the pathogenesis, li!uefaction co pletely digests the dead cells. $he end result is transfor ation of the tissue into a li!uid viscous ass. If the process #as initiated by acute infla ation *Chapter +., the aterial is fre!uently crea y yello# because of the presence of dead #hite cells and is called pus. Although gangrenous necrosis is not a distinctive pattern of cell death, the ter is still co only used in surgical clinical practice. It is usually applied to a li b, generally the lo#er leg, that has lost its blood supply and has undergone coagulation necrosis. 7hen bacterial infection is superi posed, coagulative necrosis is odified by the li!uefactive action of the bacteria and the attracted leu'ocytes *so&called !et gangrene.. Caseous necrosis, a distinctive for of coagulative necrosis, is encountered ost often in foci of tuberculous infection *Chapter D.. $he ter caseous is derived fro the cheesy #hite gross appearance of the area of necrosis *)ig. 1&+-.. :n icroscopic e"a ination, the necrotic focus appears as a orphous granular debris see ingly co posed of frag ented, coagulated cells and a orphous granular debris enclosed #ithin a distinctive infla atory border 'no#n as a granulo atous reaction *Chapter +.. Bnli'e coagulative necrosis, the tissue architecture is co pletely obliterated. Fat necrosis is a ter that is #ell fi"ed in edical parlance but does not in reality denote a specific pattern of necrosis. 6ather, it is descriptive of focal areas of fat destruction, typically occurring as a result of release of activated pancreatic lipases into the substance of the pancreas and the peritoneal cavity. $his occurs in the cala itous abdo inal e ergency 'no#n as acute pancreatitis *Chapter 1I.. In this disorder, activated pancreatic en%y es escape fro acinar cells and ducts, the activated en%y es li!uefy fat cell e branes, and the activated lipases split the triglyceride esters contained #ithin fat cells. $he released fatty acids co bine #ith calciu to produce grossly visible chal'y #hite areas *fat saponification., #hich enable the surgeon and the pathologist to identify the lesions *)ig. 1&+1.. :n histologic e"a ination, the necrosis ta'es the for of foci of shado#y outlines of necrotic fat cells, #ith basophilic calciu deposits, surrounded by an infla atory reaction.
)igure 1&1D Ische ic necrosis of the yocardiu . A, 4or al yocardiu . , Myocardiu #ith coagulation necrosis *upper t#o thirds of figure., sho#ing strongly eosinophilic anucleate yocardial fibers. Keu'ocytes in the interstitiu are an early reaction to necrotic uscle. Co pare #ith A and #ith nor al fibers in the lo#er part of the figure.

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)igure 1&1I Coagulative and li!uefactive necrosis. A, 2idney infarct e"hibiting coagulative necrosis, #ith loss of nuclei and clu ping of cytoplas but #ith preservation of basic outlines of glo erular and tubular architecture. , A focus of li!uefactive necrosis in the 'idney caused by fungal infection. $he focus is filled #ith #hite cells and cellular debris, creating a renal abscess that obliterates the nor al architecture.

Blti ately, in the living patient, ost necrotic cells and their debris disappear by a co bined process of en%y atic digestion and frag entation, follo#ed by phagocytosis of the particulate debris by leu'ocytes. If necrotic cells and cellular debris are not pro ptly destroyed and reabsorbed, they tend to attract calciu salts and other inerals and to beco e calcified. $his pheno enon, called dystrophic calcification, is considered later in the chapter.

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)igure 1&+- A tuberculous lung #ith a large area of caseous necrosis. $he caseous debris is yello#&#hite and cheesy.

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)igure 1&+1 )oci of fat necrosis #ith saponification in the esentery. $he areas of #hite chal'y deposits represent calciu soap for ation at sites of lipid brea'do#n.

E*amples of Cell In%ury and Necrosis

>aving briefly revie#ed the general causes, echanis s, and orphology of cell injury and necrotic cell death, #e no# describe so e co on for s of cell injury, na ely ische ic and hypo"ic injury, and so e types of to"ic injury. Although apoptosis contributes to cell death in so e of these conditions, it has any uni!ue features and is therefore discussed later in the chapter. IS.HE*I. A,D HYPO1I. I,60RY $his is the ost co on type of cell injury in clinical edicine and has been studied e"tensively in hu ans, in e"peri ental ani als, and in culture syste s. +A&+I 6easonable scenarios concerning the echanis s underlying the orphologic changes have e erged. >ypo"ia refers to any state of reduced o"ygen availability. It ay be caused by reduced a ounts or saturation of he oglobin. Ische ia, on the other hand, is brought about by reduced blood flo#, usually as a conse!uence of a echanical obstruction in the arterial syste but so eti es as a result of a catastrophic fall in blood pressure or loss of blood. In contrast to hypo"ia, during #hich glycolytic energy production can continue, ische ia co pro ises the delivery of substrates for glycolysis. $hus, in ische ic tissues, anaerobic energy generation stops after glycolytic substrates are e"hausted, or glycolytic function beco es inhibited by the accu ulation of etabolites that #ould have been re oved other#ise by blood flo#. )or this reason, ischemia tends to injure tissues faster than does hypoxia . Types of Ischemic In%ury. Ische ic injury is the ost co on clinical e"pression of cell injury by o"ygen deprivation. $he ost useful odels for studying ische ic injury involve co plete occlusion of one of the end&arteries to an organ *e.g., a coronary artery. and e"a ination of the tissue *e.g., cardiac uscle. in areas supplied by the artery. Co ple" pathologic changes occur in diverse cellular syste s during ische ia. Bp to a certain point, for a duration that varies a ong different types of cells, the injury is a enable to repair, and the affected cells can recover if o"ygen and etabolic substrates are again ade available by restoration of blood flo#. 7ith further e"tension of the ische ic duration, cell structure continues to deteriorate, o#ing to relentless progression of ongoing injury echanis s. 7ith ti e, the energetic achinery of the cell&the itochondrial o"idative po#erhouse and the glycolytic path#ay&beco es irreparably da aged, and restoration of blood flo# *reperfusion. cannot rescue the da aged cell. 3ven if the cellular energetic achinery #ere to re ain intact, irreparable da age to the geno e or to cellular e branes #ill ensure a lethal outco e regardless of reperfusion. $his irreversible injury is usually anifested as necrosis, but apoptosis ay also play a role. Bnder certain circu stances, #hen blood flo# is restored to cells that have been previously ade ische ic but have not died, injury is often parado"ically e"acerbated and proceeds at an accelerated pace. As a conse!uence, reperfused tissues ay sustain loss of cells i# additio# to cells that are irre$ersi&ly da%aged at the e#d of ische%ia . $his is a clinically i portant process that contributes to net tissue da age during yocardial and cerebral infarction, as described in Chapters 1+ and +D. $his so&called ische%ia2reperf sio# i#+ ry *discussed later. is particularly significant because appropriate edical treat ent can decrease the fraction of cells

that

ay other#ise be destined to die in the (area at ris'.(

Mechanisms of Ischemic Cell In%ury. $he se!uence of events follo#ing hypo"ia #as described earlier and is su ari%ed in )igure 1&++.+D,+I Briefly, as the o"ygen tension #ithin the cell decreases, there is loss of o"idative phosphorylation and decreased generation of A$P. $he depletion of A$P results in failure of the sodiu pu p, #ith loss of potassiu , influ" of sodiu and #ater, and cell s#elling. $here is progressive loss of glycogen and decreased protein synthesis. $here ay be severe functional conse!uences at this stage. )or instance, heart uscle ceases to contract #ithin C- seconds of coronary occlusion. 4ote, ho#ever, that loss of contractility does not ean cell death. If hypo"ia continues, #orsening A$P depletion causes further orphologic deterioration. $he cytos'eleton disperses, resulting in the loss of ultrastructural features such as icrovilli and the for ation of (blebs( at the cell surface *see )ig. 1&1A.. (Myelin figures,( derived fro plas a as #ell as organellar e branes, ay be seen #ithin the cytoplas or e"tracellularly. $hey are thought to result fro dissociation of lipoproteins #ith un as'ing of phosphatide groups, pro oting the upta'e and intercalation of #ater bet#een the la ellar stac's of e branes. At this ti e, the itochondria are usually s#ollen, o#ing to loss of volu e control by these organelles; the endoplas ic reticulu re ains dilated; and the entire cell is ar'edly s#ollen, #ith increased concentrations of #ater, sodiu , and chloride and a decreased concentration of potassiu . If o5yge# is restored) all of these dist r&a#ces are re$ersi&le .
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)igure 1&++ Postulated se!uence of events in reversible and irreversible ische ic cell injury. 4ote that although reduced o"idative phosphorylation and A$P levels have a central role, ische ia can cause direct e brane da age. 36, endoplas ic reticulu ; C2, creatine 'inase; KD>, lactate dehydrogenase; 64P, ribonucleoprotein.

If ische%ia persists) irre$ersi&le i#+ ry a#d #ecrosis e#s e . Irreversible injury is associated orphologically #ith severe s#elling of itochondria, e"tensive da age to plas a e branes, and s#elling of lysoso es *see )ig. 1&1A(.. Karge, flocculent, a orphous densities develop in the itochondrial atri". In the yocardiu , these are indications of irreversible injury and can be seen as early as <- to @- inutes after ische ia. Massive influ" of calciu into the cell then occurs, particularly if the ische ic %one is reperfused. Death is ainly by necrosis, but apoptosis also contributes; the apoptotic path#ay is activated probably by release of pro&apoptotic olecules fro lea'y itochondria. After death, cell co ponents are progressively degraded, and there is #idespread lea'age of cellular en%y es into the e"tracellular space and, conversely, entry of e"tracellular acro olecules fro the interstitial space into the dying cells. )inally, the dead cell ay beco e replaced by large asses co posed of phospholipids in the for of yelin figures. $hese are then either phagocytosed by other cells or degraded further into fatty acids. (alcification of such fatty acid residues ay occur #ith the for ation of calciu soaps. As #e entioned previously, lea'age of intracellular en%y es and other proteins across the abnor ally per eable plas a e brane and into the plas a provides i portant clinical para eters of cell death. )or e"a ple, elevated seru levels of cardiac uscle creatine 'inase MB and troponin are valuable clinical indicators of yocardial infarction, an area of cell death in heart uscle *Chapter 1+.. IS.HE*IA2REPER-0SIO, I,60RY 6estoration of blood flo# to ische ic tissues can result in recovery of cells if they are reversibly injured, or not affect the outco e if irreversible cell da age has occurred. >o#ever, depending on the intensity and duration of the ische ic insult, variable nu bers of cells ay proceed to die after blood flo# resu es, by necrosis as #ell as by apoptosis. <- $he affected tissues often sho# neutrophilic infiltrates. As noted earlier, this ische ia&reperfusion injury is a clinically i portant

process in such conditions as therapeutic interventions.

yocardial infarction and stro'e and

ay be a enable to
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>o# does reperfusion injury occurJ $he li'ely ans#er is that new damaging processes are set in otion during reperfusion, causing the death of cells that ight have recovered other#ise. <1,<+ 9everal echanis s have been proposed0 4e# da age ay be initiated during reo"ygenation by increased generation of oxygen free radicals fro parenchy al and endothelial cells and fro infiltrating leu'ocytes. <1,<< 9upero"ide anions can be produced in reperfused tissue as a result of inco plete and vicarious reduction of o"ygen by da aged itochondria or because of the action of o"idases derived fro leu'ocytes, endothelial cells, or parenchy al cells. <+ Cellular antio"idant defense echanis s ay also be co pro ised by ische ia, favoring the accu ulation of radicals. )ree radical scavengers ay be of therapeutic benefit. 6eactive o"ygen species can further pro ote the mitochondrial permea#ility transition , referred to earlier, #hich, #hen it occurs, precludes itochondrial energi%ation and cellular A$P recovery and leads to cell death.+, Ische ic injury is associated #ith inflammation as a result of the production of cyto'ines and increased e"pression of adhesion olecules by hypo"ic parenchy al and endothelial cells.<1,<< $hese agents recruit circulating poly orphonuclear leu'ocytes to reperfused tissue; the ensuing infla ation causes additional injury *Chapter +.. $he i portance of neutrophil influ" in reperfusion injury has been de onstrated by e"peri ental studies that have used anti&infla atory interventions, such as antibodies to cyto'ines or adhesion olecules, to reduce the e"tent of the injury.<-,<+ 6ecent data suggest that activation of the complement pathway ay contribute to ische ia&reperfusion injury.<@ $he co ple ent syste is involved in host defense and is an i portant echanis of i une injury *Chapter C.. 9o e IgM antibodies have a propensity to deposit in ische ic tissues, for un'no#n reasons, and #hen blood flo# is resu ed, co ple ent proteins bind to the antibodies, are activated, and cause cell injury and infla ation. 2noc'out ice lac'ing several co ple ent proteins are resistant to this type of injury.<,

.HE*I.AL I,60RY $he echanis s by #hich che icals, certain drugs, and to"ins produce injury are described in greater detail in Chapter I in the discussion of environ ental disease. >ere #e #ill describe t#o for s of che ically induced injury as e"a ples of the se!uence of events leading to cell death. Che icals induce cell injury by one of t#o general echanis s0 <C 9o e che icals can act directly by co bining #ith so e critical olecular co ponent or cellular organelle. )or e"a ple, in mercuric chloride poisoning, ercury binds to the sulfhydryl groups of the cell e brane and other proteins, causing increased e brane per eability and inhibition of A$Pase&dependent transport. In such instances, the greatest da age is usually to the cells that use, absorb, e"crete, or concentrate the che icals & in the case of ercuric chloride, the cells of the gastrointestinal tract and 'idney *Chapter I.. (yanide poisons itochondrial cytochro e o"idase and bloc's o"idative phosphorylation. Many antineoplastic che otherapeutic agents and antibiotic drugs also induce cell da age by direct cytoto"ic effects. Most other che icals are not biologically active but ust be converted to reactive to"ic etabolites, #hich then act on target cells. $his odification is usually acco plished by the P&@,- i"ed function o"idases in the s ooth endoplas ic reticulu of the liver and other organs.<A,<D Although these etabolites ight cause e brane da age and cell

injury by direct covalent #inding to e brane protein and lipids, by far the ost i portant echanis of e brane injury involves the for ation of reactive free radicals and subse!uent lipid pero"idation.

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)igure 1&+< 9e!uence of events leading to fatty change and cell necrosis in carbon tetrachloride *CCl@. to"icity. 636, rough endoplas ic reticulu ; 936, s ooth endoplas ic reticulu .

$he diverse echanis s of che ical injury are #ell illustrated by carbon tetrachloride and aceta inophen . (ar#on tetrachloride *CCl@. #as once used #idely in the dry&cleaning industry. <I $he to"ic effect of CCl @ is due to its conversion by P&@,- to the highly reactive toxic free radical CCl< *CCl@ N e ? CCl< N Cl&. *)ig. 1&+<.. $he free radicals produced locally cause autoo"idation of the polyenoic fatty acids present #ithin the e brane phospholipids. $here, o"idative deco position of the lipid is initiated, and organic pero"ides are for ed after reacting #ith o"ygen *lipid pero"idation.. $his reaction is autocatalytic in that ne# radicals are for ed fro the pero"ide radicals the selves. $hus, rapid brea'do#n of the structure and function of the endoplas ic reticulu is due to deco position of the lipid. It is no surprise, therefore, that ((l@&induced liver cell injury is #oth severe and extremely rapid in onset . 7ithin less than <- inutes, there is a decline in hepatic protein synthesis; #ithin + hours, there is s#elling of s ooth endoplas ic reticulu and dissociation of riboso es fro the rough endoplas ic reticulu . Kipid e"port fro the hepatocytes is reduced o#ing to their inability to synthesi%e apoprotein to co ple" #ith triglycerides and thereby facilitate lipoprotein secretion. $he result is the fatty liver of CCl @ poisoning *)ig. 1&+@.. *)atty liver is discussed later in the chapter.. Mitochondrial injury then occurs, and this is follo#ed by progressive s#elling of the cells due to increased per eability of the plas a e brane. Plas a e brane da age is thought to be caused by relatively stable fatty aldehydes, #hich are produced by lipid pero"idation in the s ooth endoplas ic reticulu but are able to act at distant sites. $his is follo#ed by assive influ" of calciu and cell death *see )ig. 1&+<..
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)igure 1&+@ 6at liver cell @ hours after carbon tetrachloride into"ication, #ith s#elling of endoplas ic reticulu and shedding of riboso es. At this stage, itochondria are unaltered. *Courtesy of Dr. :. Iseri, Bniversity of Maryland, Balti ore, MD..

Acetaminophen (+ylenol), a co only used analgesic drug, is deto"ified in the liver through sulfation and glucuronidation, and s all a ounts are converted by cytochro e P&@,-&cataly%ed o"idation to an electrophilic, highly to"ic etabolite. @- $his etabolite itself is deto"ified by interaction #ith 19>. 7hen large doses of the drug are ingested, 19> is depleted, and thus the to"ic etabolites accu ulate in the cell, destroy nucleophilic acro olecules, and covalently bind proteins and nucleic acids. $he decrease in 19> concentration, coupled #ith covalent binding of to"ic etabolites, increases drug to"icity, resulting in assive liver cell necrosis, usually < to , days after the ingestion of to"ic doses. $his hepatoto"icity correlates #ith lipid pero"idation and can be reduced by ad inistration of antio"idants, suggesting that the o"idative da age ay be ore i portant than covalent binding in the ulti ate to"icity of the drug.

poptosis
Apoptosis is a path#ay of cell death that is induced by a tightly regulated intracellular progra in #hich cells destined to die activate en%y es that degrade the cells/ o#n nuclear D4A and nuclear

and cytoplas ic proteins. $he cell/s plas a e brane re ains intact, but its structure is altered in such a #ay that the apoptotic cell beco es an avid target for phagocytosis. $he dead cell is rapidly cleared, before its contents have lea'ed out, and therefore cell death by this path#ay does not elicit an infla atory reaction in the host. $hus, apoptosis is funda entally different fro necrosis, #hich is characteri%ed by loss of e brane integrity, en%y atic digestion of cells, and fre!uently a host reaction *see )ig. 1&I and $able 1&+.. >o#ever, apoptosis and necrosis so eti es coe"ist, and they ay share so e co on features and echanis s. .A0SES O- APOPTOSIS Apoptosis #as initially recogni%ed in 1IA+ by its distinctive orphology and na ed after the 1ree' designation for (falling off.( @+ It occurs nor ally in any situations, and serves to eli inate un#anted or potentially har ful cells and cells that have outlived their usefulness. It is also a pathologic event #hen cells are da aged beyond repair, especially #hen the da age affects the cell/s D4A; in these situations, the irreparably da aged cell is eli inated. Apoptosis is responsible for nu erous physiologic, adaptive, and pathologic events, listed ne"t. Apoptosis i# Physiologic Sit atio#s Death by apoptosis is a nor al pheno enon that serves to eli inate cells that are no longer needed, as, for e"a ple, during develop ent, and to aintain a steady nu ber of various cell populations in tissues. It is i portant in the follo#ing physiologic situations0 The progra%%ed destr ctio# of cells d ri#g e%&ryoge#esis , including i plantation, organogenesis, develop ental involution, and eta orphosis. $he ter (progra ed cell death( #as originally coined to denote death of specific cell types at defined ti es during develop ent.@< Apoptosis is a generic ter for this pattern of cell death, regardless of the conte"t, but it is often used interchangeably #ith (progra ed cell death.( Hor%o#e2depe#de#t i#$ol tio# i# the ad lt, such as endo etrial cell brea'do#n during the enstrual cycle, ovarian follicular atresia in the enopause, the regression of the lactating breast after #eaning, and prostatic atrophy after castration. .ell deletio# i# proliferati#g cell pop latio#s , such as intestinal crypt epithelia, in order to aintain a constant nu ber. Death of host cells that have served their useful purpose, such as neutrophils in an ac te i#fla%%atory respo#se, and ly phocytes at the end of an immune response. In these situations, cells undergo apoptosis because they are deprived of necessary survival signals, such as gro#th factors. Eli%i#atio# of pote#tially har%f l self2reacti$e ly%phocytes , either before or after they have co pleted their aturation *Chapter C.. .ell death i#d ced &y cytoto5ic T cells , a defense echanis against viruses and tu ors that serves to eli inate virus&infected and neoplastic cells. $he sa e echanis is responsible for cellular rejection of transplants *Chapter C..

Apoptosis i# Pathologic .o#ditio#s Death by apoptosis is also responsible for loss of cells in a variety of pathologic states0 .ell death prod ced &y a $ariety of i#+ rio s sti% li . )or instance, radiation and cytoto"ic anticancer drugs da age D4A, and if repair echanis s cannot cope #ith the injury the cell 'ills itself by apoptosis. In these situations, eli ination of the cell ay be a better alternative than ris'ing utations and translocations in the da aged D4A, #hich ay result in alignant transfor ation. $hese injurious sti uli, as #ell as heat and hypo"ia, can induce apoptosis if the insult is ild, but large doses of the sa e sti uli result in necrotic cell death. 3ndoplas ic reticulu *36. stress, #hich is induced by the accu ulation of unfolded proteins, also triggers apoptotic death of cells *described later in the chapter.. .ell i#+ ry i# certai# $iral diseases , such as viral hepatitis, in #hich loss of infected cells

is largely because of apoptotic death. Pathologic atrophy i# pare#chy%al orga#s after d ct o&str ctio# , such as occurs in the pancreas, parotid gland, and 'idney. .ell death i# t %ors, ost fre!uently during regression but also in actively gro#ing tu ors. As #e entioned earlier, even in situations in #hich cell death is ainly by necrosis, the path#ay of apoptosis ay contribute. )or instance, injurious sti uli that cause increased itochondrial per eability trigger apoptosis. orphologic and bioche ical

Before the echanis s of apoptosis are discussed, #e describe the characteristics of this process.

Morphology. $he follo#ing orphologic features, so e best seen #ith the electron icroscope, characteri%e cells undergoing apoptosis *)ig. 1&+,.. Cell shrin3age. $he cell is s aller in si%e; the cytoplas is dense; and the organelles, although relatively nor al, are ore tightly pac'ed. Chromatin condensation. $his is the ost characteristic feature of apoptosis. $he chro atin aggregates peripherally, under the nuclear e brane, into dense asses of various shapes and si%es. $he nucleus itself ay brea' up, producing t#o or ore frag ents. Formation of cytoplasmic &le&s and apoptotic &odies. $he apoptotic cell first sho#s e"tensive surface blebbing, then undergoes frag entation into e brane&bound apoptotic bodies co posed of cytoplas and tightly pac'ed organelles, #ith or #ithout nuclear frag ents. Phagocytosis of apoptotic cells or cell &odies, usually &y macrophages. $he apoptotic bodies are rapidly degraded #ithin lysoso es, and the adjacent healthy cells igrate or proliferate to replace the space occupied by the no# deleted apoptotic cell.

Plas a e branes are thought to re ain intact during apoptosis, until the last stages, #hen they beco e per eable to nor ally retained solutes. $his classical description is accurate #ith respect to apoptosis during physiologic conditions such as e bryogenesis and deletion of i une cells. >o#ever, for s of cell death #ith features of necrosis as #ell as of apoptosis are not unco on after injurious sti uli.@@ Bnder such conditions, the severity, rather than the specificity, of sti ulus deter ines the for in #hich death is e"pressed. If necrotic features are predo inant, early plas a e brane da age occurs, and cell s#elling, rather than shrin'age, is seen. :n histologic e"a ination, in tissues stained #ith he ato"ylin and eosin, apoptosis involves single cells or s all clusters of cells. $he apoptotic cell appears as a round or oval ass of intensely eosinophilic cytoplas #ith dense nuclear chro atin frag ents *)ig. 1&+C.. Because the cell shrin'age and for ation of apoptotic bodies are rapid and the frag ents are !uic'ly phagocytosed, considerable apoptosis ay occur in tissues before it beco es apparent in histologic sections. In addition, apoptosis&in contrast to necrosis& does not elicit infla ation, a'ing it ore difficult to detect histologically. (IO.HE*I.AL -EAT0RES O- APOPTOSIS

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)igure 1&+, Bltrastructural features of apoptosis. 9o e nuclear frag ents sho# peripheral crescents of co pacted chro atin, #hereas others are unifor ly dense. *)ro 2err R)6, >ar on B50 Definition and incidence of apoptosis0 a historical perspective. In $o ei KD, Cope ): *eds.0 Apoptosis0 $he Molecular Basis of Cell Death. Cold 9pring >arbor, 48, Cold 9pring >arbor Kaboratory Press, 1II1, pp ,&+I..

Apoptotic cells usually e"hibit a distinctive constellation of bioche ical odifications that underlie the structural changes described above. 9o e of these features ay be seen in necrotic cells also, but other alterations are ore specific. Protein Clea(age. A specific feature of apoptosis is protein hydrolysis involving the activation of several e bers of a fa ily of cysteine proteases na ed caspases.@, Many caspases are present in nor al cells as inactive pro&en%y es, and they need to be activated to induce apoptosis. Active caspases cleave any vital cellular proteins, such as la ins, and thus brea' up the nuclear scaffold and cytos'eleton; in addition, caspases activate D4Ases, #hich degrade nuclear D4A. $hese changes underlie the nuclear and cytoplas ic structural alterations seen in apoptotic cells. DN 5rea3do!n. Apoptotic cells e"hibit a characteristic brea'do#n of D4A into large ,-& to <--& 'ilobase pieces.@C 9ubse!uently, there is internucleoso al cleavage of D4A into oligonucleoso es, in ultiples of 1D- to +-- base pairs, by Ca +N& and Mg+N&dependent endonucleases. $he frag ents ay be visuali%ed by agarose gel electrophoresis as D4A ladders *)ig. 1&+A.. 3ndonuclease activity also for s the basis for detecting cell death by cytoche ical techni!ues that recogni%e double&stranded brea's of D4A. @A >o#ever, internucleoso al D4A cleavage is not specific for apoptosis. A (s eared( pattern of D4A frag entation is thought to be indicative of necrosis, but this ay be a late autolytic pheno enon, and typical D4A ladders ay be seen in necrotic cells as #ell.@A
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)igure 1&+C A, Apoptosis of epider al cells in an i une& ediated reaction. $he apoptotic cells are visible in the epider is #ith intensely eosinophilic cytoplas and s all, dense nuclei. >S3 stain. *Courtesy of Dr. 9cott 1ranter, Brigha and 7o en/s >ospital, Boston, MA.. , >igh po#er of apoptotic cell in liver in i une& ediated hepatic cell injury. *Courtesy of Dr. Dhanpat Rain, 8ale Bniversity, 4e# >aven, C$..

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)igure 1&+A Agarose gel electrophoresis of D4A e"tracted fro culture cells. 3thidiu bro ide stain; photographed under ultraviolet illu ination. $ane A, Control culture. $ane , Culture of cells e"posed to heat sho#ing e"tensive apoptosis; note ladder pattern of D4A frag ents, #hich represent ultiples of oligonucleoso es. $ane (, Culture sho#ing assive necrosis; note diffuse s earing of D4A. $he ladder pattern is produced by en%y atic cleavage of nuclear D4A into nucleoso e&si%ed frag ents, usually ultiples of 1D-&+-- base pairs. +hese patterns are characteristic of #ut not specific for apoptosis and necrosis, respectively. *)ro 2err R)6, >ar on B50 Definition and incidence of apoptosis0 a historical perspective. In $o ei KD, Cope ): FedsG0 Apoptosis0 $he Molecular Basis of Cell Death. Cold 9pring >arbor, 48, Cold 9pring >arbor Kaboratory Press, 1II1, p 1<..

Phagocytic 'ecognition. Apoptotic cells e"press phosphatidylserine in the outer layers of their plas a e branes, the phospholipid having (flipped( out fro the inner layers. *Because of these changes, apoptotic cells can be identified by binding of special dyes, such as Anne"in 5.. In so e types of apoptosis, thro bospondin, an adhesive glycoprotein, is also e"pressed on the surfaces of apoptotic bodies, and other proteins secreted by phagocytes ay bind to apoptotic cells and opsoni%e the cells for phagocytosis.@D $hese alterations per it the early recognition of dead cells by acrophages, resulting in phagocytosis #ithout the release of proinfla atory cellular

co ponents.@I In this #ay, the apoptotic response disposes of cells #ith the surrounding tissue. *E.HA,IS*S O- APOPTOSIS

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Apoptosis is induced by a cascade of olecular events that ay be initiated in distinct #ays and cul inate in the activation of caspases *)ig. 1&+D..@, Because too uch or too little apoptosis is thought to underlie any diseases, such as degenerative diseases and cancer, there is great interest in elucidating the echanis s of this for of cell death. $re endous progress has been ade in our understanding of apoptosis. :ne of the re ar'able facts to e erge is that the basic echanis s of apoptosis are conserved in all eta%oans. ,- In fact, so e of the ajor brea'throughs ca e fro observations ade in the ne atode (aenorha#ditis elegans, #hose develop ent proceeds by a highly reproducible, progra ed pattern of cell gro#th follo#ed by cell death. 9tudies of utant #or s have allo#ed the identification of specific genes *called ced genes, for cell death abnor al. that initiate or inhibit apoptosis and for #hich there are defined a alian ho ologues. $he process of apoptosis ay be divided into an initiation phase, during #hich caspases beco e catalytically active, and an e"ecution phase, during #hich these en%y es act to cause cell death. Initiation of apoptosis occurs principally by signals fro t#o distinct but convergent path#ays & the e"trinsic, or receptor&initiated, path#ay and the intrinsic, or itochondrial, path#ay. Both path#ays converge to activate caspases. 7e #ill describe these t#o path#ays separately because they involve largely distinct olecular interactions, but it is i portant to re e ber that they ay be interconnected at nu erous steps.
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)igure 1&+D Mechanis s of apoptosis. Kabeled *1. are so e of the ajor inducers of apoptosis. $hese include specific death ligands *tu or necrosis factor F$4)G and )as ligand., #ithdra#al of gro#th factors or hor ones, and injurious agents *e.g., radiation.. 9o e sti uli *such as cytoto"ic cells. directly activate e"ecution caspases (right). :thers act by #ay of adapter proteins and initiator caspases, or by itochondrial events involving cytochro e c. *+. Control and regulation are influenced by e bers of the Bcl&+ fa ily of proteins, #hich can either inhibit or pro ote the cell/s death. *<. 3"ecutioner caspases activate latent cytoplas ic endonucleases and proteases that degrade nuclear and cytos'eletal proteins. $his results in a cascade of intracellular degradation, including frag entation of nuclear chro atin and brea'do#n of the cytos'eleton. *@. $he end result is for ation of apoptotic bodies containing intracellular organelles and other cytosolic co ponents; these bodies also e"press ne# ligands for binding and upta'e by phagocytic cells.

The E*trinsic 1Death 'eceptor#Initiated2 Path!ay. $his path#ay is initiated by engage ent of cell surface death receptors on a variety of cells. ,1 Death receptors are e bers of the tu or necrosis factor receptor fa ily that contain a cytoplas ic do ain involved in protein&protein interactions that is called the death domain because it is essential for delivering apoptotic signals. *9o e $4) receptor fa ily e bers do not contain cytoplas ic death do ains; their role in triggering apoptosis is uch less established.. $he best&'no#n death receptors are the type 1 $4) receptor *$4)61. and a related protein called )as *CDI,., but several others have been described. $he echanis of apoptosis induced by these death receptors is #ell illustrated by )as *)ig. 1&+I.. 7hen )as is cross&lin'ed by its ligand, e brane&bound )as ligand *)asK., three or ore olecules of )as co e together, and their cytoplas ic death do ains for a binding site for an adapter protein that also contains a death do ain and is called )ADD * ,as&associated death do ain.. )ADD that is attached to the death receptors in turn binds an inactive for of caspase&D *and, in hu ans, caspase&1-., again via a death do ain. Multiple pro&caspase&D olecules are thus brought into pro"i ity, and they cleave one another to generate active caspase&D. $he en%y e then triggers a cascade of caspase activation by cleaving and thereby activating other

pro&caspases, and the active en%y es ediate the e"ecution phase of apoptosis *discussed belo#.. $his path#ay of apoptosis can be inhibited by a protein called )KIP, #hich binds to pro& caspase&D but cannot cleave and activate the en%y e because it lac's en%y atic activity. ,+ 9o e viruses and nor al cells produce )KIP and use this inhibitor to protect infected and nor al cells fro )as& ediated apoptosis. $he sphingolipid cera ide has been i plicated as an inter ediate bet#een death receptors and caspase activation, but the role of this path#ay is unclear and re ains controversial.,<
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)igure 1&+I $he e"trinsic *death receptor&initiated. path#ay of apoptosis, illustrated by the events follo#ing )as engage ent *see te"t..

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)igure 1&<- $he intrinsic * itochondrial. path#ay of apoptosis. Death agonists cause changes in the inner itochondrial e brane, resulting in the itochondrial per eability transition *MP$. and release of cytochro e c and other pro&apoptotic proteins into the cytosol, #hich activate caspases *see te"t..

The Intrinsic 1Mitochondrial2 Path!ay. $his path#ay of apoptosis is the result of increased itochondrial per eability and release of pro&apoptotic olecules into the cytoplas , #ithout a role for death receptors.,@,,, 1ro#th factors and other survival signals sti ulate the production of anti&apoptotic e bers of the Bcl&+ fa ily of proteins. ,C $his fa ily is na ed after Bcl&+, #hich #as identified as an oncogene in a B cell ly pho a and is ho ologous to the (. elegans protein, Ced&I. $here are ore than +- proteins in this fa ily, all of #hich function to regulate apoptosis; the t#o ain anti&apoptotic ones are Bcl&+ and Bcl&". $hese anti&apoptotic proteins nor ally reside in itochondrial e branes and the cytoplas . 7hen cells are deprived of survival signals or subjected to stress, Bcl&+ andPor Bcl&" are lost fro the itochondrial e brane and are replaced by pro&apoptotic e bers of the fa ily, such as Ba', Ba", and Bi . 7hen Bcl&+PBcl&" levels decrease, the per eability of the itochondrial e brane increases, and several proteins that can activate the caspase cascade lea' out *)ig. 1&<-.. :ne of these proteins is cytochro e c, #ell 'no#n for its role in itochondrial respiration. In the cytosol, cytochro e c binds to a protein called Apaf&1 *apoptosis activating factor&1, ho ologous to Ced&@ in (. elegans., and the co ple" activates caspase&I.,A *Bcl&+ and Bcl&" ay also directly inhibit Apaf&1 activation, and their loss fro cells ay per it activation of Apaf&1.. :ther itochondrial proteins, such as apoptosis inducing factor *AI)., enter the cytoplas , #here they bind to and neutrali%e various inhibitors of apoptosis, #hose nor al function is to bloc' caspase activation. ,D $he net result is the initiation of a caspase cascade. $hus, the essence of this intrinsic pathway is a #alance #etween pro& apoptotic and protective molecules that regulate mitochondrial permea#ility and the release of death inducers that are normally se%uestered within the mitochondria . $here is !uite a lot of evidence that the intrinsic path#ay of apoptosis can be triggered #ithout a role for itochondria.,I Apoptosis ay be initiated by caspase activation upstrea of itochondria, and the subse!uent increase in itochondrial per eability and release of pro&apoptotic olecules a plify the death signal.@C >o#ever, these path#ays of apoptosis involving itochondria& independent initiation are not #ell defined. 7e have described the e"trinsic and intrinsic path#ays for initiating apoptosis as distinct, but there ay be overlaps bet#een the . )or instance, in hepatocytes, )as signaling activates a pro&apoptotic e ber of the Bcl fa ily called Bid, #hich then activates the itochondrial path#ay. It is not 'no#n if such cooperative interactions bet#een apoptosis path#ays are active in ost other cell types.
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The E*ecution Phase. $his final phase of apoptosis is ediated by a proteolytic cascade, to#ard #hich the various initiating echanis s converge. $he proteases that ediate the e"ecution phase are highly conserved across species and belong to the caspase fa ily, as previously entioned. $hey are a alian ho ologues of the ced&- gene in (. elegans.@@ $he ter caspase is based on t#o properties of this fa ily of en%y es0 the (c( refers to a cysteine protease *i.e., an en%y e #ith cysteine in its active site., and (aspase( refers to the uni!ue ability of these en%y es to cleave after aspartic acid residues. C- $he caspase fa ily, no# including ore than 1e bers, can be divided functionally into t#o basic groups&initiator and e"ecutioner&depending on the order in #hich they are activated during apoptosis. C- Initiator caspases, as #e have seen, include caspase&D and caspase&I. 9everal caspases, including caspase&< and caspase&C, serve as e"ecutioners. Ki'e any proteases, caspases e"ist as inactive pro&en%y es, or %y ogens, and ust undergo an activating cleavage for apoptosis to be initiated. Caspases have their o#n cleavage sites that can be hydroly%ed not only by other caspases but also autocatalytically. After an initiator caspase is cleaved to generate its active for , the en%y atic death progra is set in otion by rapid and se!uential activation of other caspases. 3"ecutioner caspases act on any cellular co ponents. $hey cleave cytos'eletal and nuclear atri" proteins and thus disrupt the cytos'eleton and lead to brea'do#n of the nucleus.C- In the nucleus, the targets of caspase activation include proteins involved in transcription, D4A replication, and D4A repair. In particular, caspase&< activation converts a cytoplas ic D4ase into an active for by cleaving an inhibitor of the en%y e; this D4ase induces the characteristic internucleoso al cleavage of D4A, described earlier. 'emo(al of Dead Cells. At early stages of apoptosis, dying cells secrete soluble factors that recruit phagocytes.C1 $his facilitates pro pt clearance of apoptotic cells before they undergo secondary necrosis and release their cellular contents *#hich can result in infla ation.. As already alluded to, apoptotic cells and their frag ents have ar'er olecules on their surfaces, #hich facilitates early recognition by adjacent cells or phagocytes for phagocytic upta'e and disposal. 4u erous acrophage receptors have been sho#n to be involved in the binding and engulf ent of apoptotic cells. In addition, acrophages can also secrete substances that bind specifically to apoptotic but not live cells and opsoni%e these cells for phagocytosis. In contrast to ar'ers on apoptotic cells, viable cells appear to prevent their o#n engulf ent by acrophages through e"pression of certain surface olecules *such as CD<1.. $his process of phagocytosis of apoptotic cells is so efficient that dead cells disappear #ithout leaving a trace, and infla ation is virtually absent. E1A*PLES O- APOPTOSIS $he signals that induce apoptosis include lac" of growth factor or hormone, specific engagement of death receptors, and particular injurious agents. Although the classic e"a ple of apoptosis has been progra ed death of cells during e bryogenesis, #e still do not 'no# #hat triggers apoptosis in this situation. >o#ever, any other #ell&defined e"a ples of apoptosis are 'no#n. poptosis fter Gro!th Factor Depri(ation. >or one&sensitive cells deprived of the relevant hor one, ly phocytes that are not sti ulated by antigens and cyto'ines, and neurons deprived of nerve gro#th factor die by apoptosis.C+ In all these situations, apoptosis is triggered by the intrinsic * itochondrial. path#ay and is attributable to an e"cess of pro&apoptotic e bers of the Bcl fa ily relative to anti&apoptotic e bers. DN Damage#Mediated poptosis. 3"posure of cells to radiation or che otherapeutic agents induces apoptosis by a echanis that is initiated by D4A da age *genoto"ic stress. and that involves the tu or&suppressor gene p.-.C< p,< accu ulates #hen D4A is da aged and arrests the cell cycle *at the 11 phase. to allo# ti e for repair *Chapter A.. >o#ever, if the D4A repair process fails, p,< triggers apoptosis. 7hen p,< is utated or absent *as it is in certain cancers., it is incapable of inducing apoptosis and it favors cell survival. $hus, p,< see s to serve as a critical (life or death( s#itch in the case of genoto"ic stress. $he echanis by #hich p,< triggers the

distal death effector achinery&the caspases&is co ple" but see s to involve its #ell& characteri%ed function in transcriptional activation. A ong the proteins #hose production is sti ulated by p,< are several pro&apoptotic e bers of the Bcl fa ily, notably Ba" and Ba', as #ell as Apaf&1, entioned earlier. $hese proteins activate caspases and cause apoptosis. poptosis Induced &y Tumor Necrosis Factor Family of 'eceptors. As discussed above, the cell surface receptor )as *CDI,. induces apoptosis #hen it is engaged by )as ligand *)asK or CDI,K., #hich is produced by cells of the i une syste . $his syste is i portant in the eli ination of ly phocytes that recogni%e self&antigens, and utations in )as or )asK result in autoi une diseases in hu ans and ice *Chapter C..C@ $he cyto'ine $4) is an i portant ediator of the infla atory reaction *Chapter +., but it is also capable of inducing apoptosis. *$he na e (tu or necrosis factor( arose not because the cyto'ine 'ills tu or cells directly, but because it induces thro bosis of tu or blood vessels, resulting in ische ic death of the tu or.. $he binding of $4) to $4)61 leads to association of the receptor #ith the adapter protein $6ADD *+4) receptor&associated death do ain containing protein.. $6ADD in turn binds to )ADD and leads to apoptosis through caspase activation, as #ith )as& )asK interactions.,+ $he ajor functions of $4), ho#ever, are ediated not by inducing apoptosis but by activation of the i portant transcription factor nuclear factor&TB *4)&TB.. $4)& ediated signals acco plish this by sti ulating degradation of the inhibitor of 4)&TB *ITB.. C, $he 4)&TBPITB transcriptional regulatory syste is i portant for cell survival and, as #e shall see in Chapter +, for a nu ber of infla atory responses. 9ince $4) can induce cell death and pro ote cell survival, #hat deter ines this yin and yang of its actionJ $he ans#er is unclear, but it probably depends on #hich adapter protein attaches to the $4) receptor after binding of the cyto'ine. $6ADD and )ADD favor apoptosis, and other adapter proteins, called $6A)s *$4) receptor associated factors. favor 4)&TB activation and survival. Cytoto*ic T#4ymphocyte#Mediated poptosis. Cytoto"ic $ ly phocytes *C$Ks. recogni%e foreign antigens presented on the surface of infected host cells *Chapter C.. :n recognition, C$Ks secrete perforin, a trans e brane pore&for ing olecule, #hich allo#s entry of the C$K granule serine protease called granzyme . 1ran%y e B has the ability to cleave proteins at aspartate residues and is able to activate a variety of cellular caspases. CC )n this way, the (+$ "ills target cells through #ypassing the upstream signaling events and directly induces the effector phase of apoptosis. C$Ks also e"press )asK on their surfaces and 'ill target cells by ligation of )as receptors, as described earlier.
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/ysregulated apoptosis (0too little or too much0) has been postulated to e"plain co ponents of a #ide range of diseases.CA In essence, t#o groups of disorders ay result fro such dysregulation0 Disorders associated with defective apoptosis and increased cell survival . >ere, an inappropriately lo# rate of apoptosis ay prolong the survival or reduce the turnover of abnor al cells. $hese accu ulated cells can give rise to0 *1. cancers, especially tu ors #ith p.- utations, or hor one&dependent tu ors, such as breast, prostate, or ovarian cancers *Chapter A.; and *+. autoimmune disorders, #hich could arise if autoreactive ly phocytes are not eli inated after encounter #ith self antigens *Chapter C.. /isorders associated with increased apoptosis and excessive cell death . $hese diseases are characteri%ed by a ar'ed loss of nor al or protective cells and include0 *1. neurodegenerative diseases, anifested by loss of specific sets of neurons, such as in the spinal uscular atrophies *Chapter +A.; *+. ischemic injury, as in yocardial infarction *Chapter 1+. and stro'e *Chapter +D.; and *<. death of virus&infected cells , in any viral infections *Chapter D..

$u&cellular 'esponses to In%ury

$o this point in the chapter, the focus has been on the cell as a unit. Certain conditions, ho#ever, are associated #ith distinctive alterations in cell organelles or the cytos'eleton. 9o e of these alterations coe"ist #ith those described for acute lethal injury; others represent ore chronic for s of cell injury; still others are adaptive responses that serve to aintain ho eostasis. >ere #e touch on only so e of the ore co on or interesting of these reactions. LYSOSO*AL .ATA(OLIS* Primary lysosomes are e brane&bound intracellular organelles that contain a variety of hydrolytic en%y es, including acid phosphatase, glucuronidase, sulfatase, ribonuclease, and collagenase . $hese en%y es are synthesi%ed in the rough endoplas ic reticulu and then pac'aged into vesicles in the 1olgi apparatus. Pri ary lysoso es fuse #ith e brane&bound vacuoles that contain aterial to be digested, for ing secondary lysosomes or phagolysosomes. Kysoso es are involved in the brea'do#n of phagocytosed aterial in one of t#o #ays0 heterophagy and autophagy *)ig. 1&<1.. .eterophagy. >eterophagy is the process of lysoso al digestion of aterials ingested fro the e"tracellular environ ent. 3"tracellular aterials are ta'en up by cells through the general process of endocytosis. Bpta'e of particulate atter is 'no#n as phagocytosis; upta'e of soluble s aller acro olecules is called pinocytosis. 3"tracellular aterials are endocytosed into vacuoles *endoso es or phagoso es., #hich eventually fuse #ith lysoso es to for phagolysoso es, #here the engulfed aterial is digested. >eterophagy is ost co on in the (professional( phagocytes, such as neutrophils and acrophages, although it ay also occur in other cell types. 3"a ples of heterophagocytosis include the upta'e and digestion of bacteria by neutrophils and the re oval of apoptotic cells by acrophages. utophagy. Autophagy refers to lysoso al digestion of the cell/s o#n co ponents. In this process, intracellular organelles and portions of cytosol are first se!uestered fro the cytoplas in an autophagic vacuole for ed fro riboso e&free regions of the rough endoplas ic reticulu . $he vacuole fuses #ith lysoso es or 1olgi ele ents to for an autophagolysosome.CD,CI Autophagy is a co on pheno enon involved in the re oval of da aged organelles during cell injury and the cellular re odeling of differentiation, and it is particularly pronounced in cells undergoing atrophy induced by nutrient deprivation or hor onal involution.

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)igure 1&<1 A, 9che atic representation of heterophagy (left) and autophagy (right). *6edra#n fro )a#cett D70 A $e"tboo' of >istology, 11th ed. Philadelphia, 7B 9aunders, 1IDC, p 1A.. , 3lectron icrograph of an autophagolysoso e containing a degenerating itochondrion and a orphous aterial.

$he en%y es in lysoso es are capable of degrading ost proteins and carbohydrates, but so e lipids re ain undigested. Kysoso es #ith undigested debris ay persist #ithin cells as residual #odies or ay be e"truded. $ipofuscin pigment granules represent undigested aterial derived fro intracellular lipid pero"idation. Certain indigestible pig ents, such as carbon particles inhaled fro the at osphere or inoculated pig ent in tattoos, can persist in phagolysoso es of acrophages for decades. Kysoso es are also repositories #here cells se!uester abnor al substances that cannot be

co pletely etaboli%ed. >ereditary lysosomal storage disorders, caused by deficiencies of en%y es that degrade various acro olecules, result in the accu ulation of abnor al a ounts of these co pounds in the lysoso es of cells all over the body, particularly neurons, leading to severe abnor alities *Chapter ,.. Certain drugs can disturb lysoso al function and cause ac%uired or drug&induced (iatrogenic) lysosomal diseases . Drugs in this group include chloro!uine , an anti alarial agent that raises the internal p> of the lysoso e, thus inactivating its en%y es. By inhibiting lysoso al en%y es, chloro!uine reduces tissue da age in infla atory reactions, #hich are ediated in part by en%y es released fro leu'ocytes; this action is the basis of the use of the drug in autoi une diseases li'e rheu atoid arthritis. $he sa e inhibition of en%y es, ho#ever, can result in abnor al accu ulation of glycogen and phospholipids in lysoso es, causing to"ic yopathy. I,D0.TIO, (HYPERTROPHY! O- S*OOTH E,DOPLAS*I. RETI.0L0* $he s ooth 36 is involved in the etabolis of various che icals, and cells e"posed to these che icals sho# hypertrophy of the 36 as an adaptive response that ay have i portant functional conse!uences. Protracted use of barbiturates leads to a state of tolerance, #ith a decrease in the effects of the drug and the need to use increasing doses. Patients are said to have (adapted( to the edication. $his adaptation is due to increased volu e *hypertrophy. of the s ooth 36 of hepatocytes, #hich etaboli%es the drug *)ig. 1&<+.. Barbiturates are odified in the liver by o"idative de ethylation, #hich involves the P&@,- i"ed function o"idase syste found in the s ooth 36. $he role of these en%y e odifications is to increase the solubility of a variety of co pounds *e.g., alcohol, steroids, eicosanoids, and carcinogens as #ell as insecticides and other environ ental pollutants. and thereby facilitate their secretion. <A Although this is often thought of as (deto"ification,( any co pounds are rendered more injurious by P&@,odification. In addition, the products for ed by this o"idative etabolis include reactive o"ygen species, #hich can cause injury of the cell. 7ith prolonged use, the barbiturates *and any other agents. sti ulate the synthesis of ore en%y es, as #ell as ore s ooth 36. In this anner, the cell is better able to odify the drugs and adapt to its altered environ ent. Cells adapted to one drug have increased capacity to etaboli%e other co pounds handled by the syste . $hus, if patients ta'ing phenobarbital for epilepsy increase their alcohol inta'e they ay have subtherapeutic levels of the antisei%ure edication because of induction of s ooth 36 in response to the alcohol. *ITO.HO,DRIAL ALTERATIO,S

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)igure 1&<+ 3lectron icrograph of liver fro phenobarbital&treated rat sho#ing ar'ed increase in s ooth endoplas ic reticulu . *)ro Rones AK, )a#cett D70 >ypertrophy of the agranular endoplas ic reticulu in ha ster liver induced by Phenobarbital . R >istoche Cytoche 1@0+1,, 1ICC. Courtesy of Dr. )a#cett..

7e have seen that itochondrial dysfunction plays an i portant role in cell injury and apoptosis. In addition, various alterations in the nu ber, si%e, and shape of itochondria occur in so e pathologic conditions. )or e"a ple, in cell hypertrophy and atrophy, there is an increase and decrease, respectively, in the nu ber of itochondria in cells. Mitochondria ay assu e e"tre ely large and abnor al shapes * ega itochondria., as can be seen in the liver in alcoholic liver disease and in certain nutritional deficiencies *)ig. 1&<<.. Abnor alities of itochondria are no# recogni%ed as the basis of any genetic diseases A- *Chapter ,.. In certain inherited etabolic diseases of s'eletal uscle, the mitochondrial myopathies, defects in itochondrial etabolis are associated #ith increased nu bers of itochondria that are often unusually large, have abnor al cristae, and contain crystalloids *Chapter +A.. In addition, certain benign tu ors found in salivary glands, thyroid, parathyroids, and 'idneys consist of cells *so eti es called (oncocytes(. #ith abundant enlarged itochondria, giving the cell a distinctly eosinophilic appearance.

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)igure 1&<< 3nlarged, abnor ally shaped itochondria fro the liver of a patient #ith alcoholic cirrhosis. 4ote also crystalline for ations in the itochondria.

.YTOS7ELETAL A(,OR*ALITIES Abnor alities of the cytos'eleton underlie a variety of pathologic states. $he cytos"eleton consists of icrotubules *+- to +, n in dia eter., thin actin fila ents *C to D n ., thic' yosin fila ents *1, n ., and various classes of inter ediate fila ents *1- n .. 9everal other nonpoly eri%ed and nonfila entous for s of contractile proteins also e"ist. Cytos'eletal abnor alities ay be reflected by0 *1. defects in cell function, such as cell loco otion and intracellular organelle ove ents, and *+. in so e instances by intracellular accu ulations of fibrillar aterial. :nly a fe# e"a ples are cited. +hin filaments. $hin fila ents are co posed of actin, yosin, and their associated regulatory proteins.A1 )unctioning thin fila ents are essential for various stages of leu'ocyte ove ent or the ability of such cells to perfor phagocytosis ade!uately. 9o e drugs and to"ins target actin fila ents and thus affect these processes. )or e"a ple, cytochalasin B prevents poly eri%ation of actin fila ents, and phalloidin, a to"in of the ushroo Amanita phalloides, also binds actin fila ents. 'icrotu#ules. Defects in the organi%ation of icrotubules can inhibit sper otility, causing ale sterility, and can i obili%e the cilia of respiratory epitheliu , causing interference #ith the ability of this epitheliu to clear inhaled bacteria, leading to bronchiectasis *2artagener/s syndro e, or the immotile cilia syndrome; Chapter 1,.. Microtubules, li'e icrofila ents, are essential for leu'ocyte igration and phagocytosis. Drugs such as colchicine bind to tubulin and prevent the asse bly of icrotubules. $he drug is used in acute attac's of gout to prevent leu'ocyte igration and phagocytosis in response to deposition of urate crystals. Microtubules are an essential co ponent of the itotic spindle, #hich is re!uired for cell division. Drugs that bind to icrotubules *e.g., vinca al'aloids. can be antiproliferative and therefore act as antitu or agents. )ntermediate filaments. $hese co ponents provide a fle"ible intracellular scaffold that organi%es the cytoplas and resists forces applied to the cell. A+ $he inter ediate fila ents are divided into five classes, including 'eratin fila ents *characteristic of epithelial cells., neurofila ents *neurons., des in fila ents * uscle cells., vi entin fila ents *connective tissue cells., and glial fila ents *astrocytes.. Accu ulations of 'eratin fila ents and neurofila ents are associated #ith certain types of cell injury. )or e"a ple, the 'allory #ody, or (alcoholic hyalin,( is an eosinophilic intracytoplas ic inclusion in liver cells that is characteristic of alcoholic liver disease,A< although it can be present in other conditions. 9uch inclusions are co posed predo inantly of "eratin inter ediate fila ents *)ig. 1&<@.. In the nervous syste , neurofila ents are present in the a"on, #here they provide structural support. $he neurofi#rillary tangle found in the brain in Al%hei er/s disease contains icrotubule&associated proteins and neurofila ents, a reflection of a disrupted neuronal cytos'eleton *Chapter +D.. Mutations in inter ediate fila ent genes cause ultiple hu an disorders, including yopathies, neurologic diseases, and s'in diseases.

Much of the e phasis on the functions of the cytos'eleton has been on its echanical role, in aintaining cellular architecture and in cell attach ent and loco otion. It has recently been appreciated that cytos"eletal proteins are lin"ed to many cellular receptors , such as ly phocyte receptors for antigens, and are active participants in signal transduction by these receptors *Chapter <.. $herefore, defects in the lin's bet#een receptors and cytos'eletal proteins ay affect any cellular responses. $he *is"ott&Aldrich syndrome is an inherited disease characteri%ed by

ec%e a, platelet abnor alities, and i une deficiency. $he protein that is utated in this disease is involved in lin'ing ly phocyte antigen receptors *and perhaps other receptors. to the cytos'eleton, and defects in the protein interfere #ith diverse cellular responses *Chapter C..A@

Intracellular

ccumulations

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)igure 1&<@ A, $he liver of alcohol abuse *chronic alcoholis .. >yaline inclusions in the hepatic parenchy al cell in the center appear as eosinophilic net#or's disposed about the nuclei (arrow). , 3lectron icrograph of alcoholic hyalin. $he aterial is co posed of inter ediate *pre'eratin. fila ents and an a orphous atri".

:ne of the anifestations of etabolic derange ents in cells is the intracellular accu ulation of abnor al a ounts of various substances. $he stoc'piled substances fall into three categories0 *1. a normal cellular constituent accu ulated in e"cess, such as #ater, lipids, proteins, and carbohydrates; *+. an a#normal su#stance, either e"ogenous, such as a ineral or products of infectious agents, or endogenous, such as a product of abnor al synthesis or etabolis ; and *<. a pigment. $hese substances ay accu ulate either transiently or per anently, and they ay be har less to the cells, but on occasion they are severely to"ic. $he substance ay be located in either the cytoplas *fre!uently #ithin phagolysoso es. or the nucleus. In so e instances, the cell ay be producing the abnor al substance, and in others it ay be erely storing products of pathologic processes occurring else#here in the body. Many processes result in abnor al intracellular accu ulations, but ost accu ulations are attributable to three types of abnor alities *)ig. 1&<,.. 1. A normal endogenous su#stance is produced at a normal or increased rate, #ut the rate of meta#olism is inade%uate to remove it . An e"a ple of this type of process is fatty change in the liver because of intracellular accu ulation of triglycerides *see later.. Another is the appearance of reabsorption protein droplets in renal tubules because of increased lea'age of protein fro the glo erulus. 2. A normal or a#normal endogenous su#stance accumulates #ecause of genetic or ac%uired defects in the meta#olism, pac"aging, transport, or secretion of these su#stances. :ne e"a ple is the group of conditions caused by genetic defects of specific en%y es involved in the etabolis of lipid and carbohydrates resulting in intracellular deposition of these substances, largely in lysoso es. $hese so&called storage diseases are discussed in Chapter ,. Another is alpha1&antitrypsin deficiency, in #hich a single a ino acid substitution in the en%y e results in defects in protein folding and accu ulation of the en%y e in the endoplas ic reticulu of the liver in the for of globular eosinophilic inclusions *see later and Chapter 1D.. <. An a#normal exogenous su#stance is deposited and accumulates because the cell has neither the en%y atic achinery to degrade the substance nor the ability to transport it to other sites. Accu ulations of carbon particles and such non etaboli%able che icals as silica particles are e"a ples of this type of alteration. *hatever the nature and origin of the intracellular accumulation, it implies the storage of some product #y individual cells. If the overload is due to a syste ic derange ent and can be brought under control, the accu ulation is reversible. In genetic storage diseases, accu ulation is

progressive, and the cells ay beco e so overloaded as to cause secondary injury, leading in so e instances to death of the tissue and the patient. LIPIDS All ajor classes of lipids can accu ulate in cells0 triglycerides, cholesterolPcholesterol esters, and phospholipids. Phospholipids are co ponents of the yelin figures found in necrotic cells. In addition, abnor al co ple"es of lipids and carbohydrates accu ulate in the lysoso al storage diseases *Chapter ,.. >ere #e concentrate on triglyceride and cholesterol accu ulations.

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)igure 1&<, Mechanis s of intracellular accu ulations0 *1. abnor al etabolis , as in fatty change in the liver; *+. utations causing alterations in protein folding and transport, as in alpha1&antitrypsin deficiency; *<. deficiency of critical en%y es that prevent brea'do#n of substrates that accu ulate in lysoso es, as in lysoso al storage diseases; and *@. inability to degrade phagocytosed particles, as in he osiderosis and carbon pig ent accu ulation.

Steatosis (-atty .ha#ge!

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$he ter s steatosis and fatty change describe abnor al accu ulations of triglycerides #ithin parenchy al cells. )atty change is often seen in the liver because it is the ajor organ involved in fat etabolis , but it also occurs in heart, uscle, and 'idney. $he causes of steatosis include to"ins, protein alnutrition, diabetes ellitus, obesity, and ano"ia. )n industrialized nations, #y far the most common cause of significant fatty change in the liver (fatty liver) is alcohol a#use *Chapter 1D..A, Different echanis s account for triglyceride accu ulation in the liver. )ree fatty acids fro adipose tissue or ingested food are nor ally transported into hepatocytes. In the liver, they are esterified to triglycerides, converted into cholesterol or phospholipids, or o"idi%ed to 'etone bodies. 9o e fatty acids are synthesi%ed fro acetate as #ell. 6elease of triglycerides fro the hepatocytes re!uires association #ith apoproteins to for lipoproteins, #hich ay then traverse the circulation *Chapter @.. 1xcess accumulation of triglycerides within the liver may result from defects in any one of the events in the se%uence from fatty acid entry to lipoprotein exit *)ig. 1& <CA.. A nu ber of such defects are induced by alcohol, a hepatoto"in that alters itochondrial and icroso al functions. CCl@ and protein alnutrition act by decreasing synthesis of apoproteins. Ano"ia inhibits fatty acid o"idation. 9tarvation increases fatty acid obili%ation fro the peripheral stores. $he significance of fatty change depends on the cause and severity of the accu ulation. 7hen ild, it ay have no effect on cellular function. More severe fatty change ay i pair cellular function, typically #hen so e vital intracellular process is also i paired *e.g., in CCl @ poisoning.. As a severe for of injury, fatty change ay be a harbinger of cell death. In recent years, nonalcoholic steatohepatitis and nonalcoholic fatty liver disease have been recogni%ed as fairly co on disease entities that ay lead to cirrhosis and even hepatocellular cancer *Chapter 1D.. Morphology. )atty change is ost often seen in the liver and heart. In all organs, fatty change appears as clear vacuoles #ithin parenchy al cells. Intracellular accu ulations of #ater or polysaccharides *e.g., glycogen. ay also produce clear vacuoles, and it beco es necessary to resort to special techni!ues to distinguish these three types of clear vacuoles. $he identification of lipids re!uires the avoidance of fat solvents co only used in paraffin e bedding for routine he ato"ylin and eosin stains. $o identify the fat, it is necessary to prepare fro%en tissue sections of either fresh or a!ueous for alin&fi"ed tissues. $he sections ay then be stained #ith 9udan I5 or :il

6ed&:, both of #hich i part an orange&red color to the contained lipids. $he periodic acid&9chiff *PA9. reaction is co only e ployed to identify glycogen, although it is by no eans specific. 7hen neither fat nor polysaccharide can be de onstrated #ithin a clear vacuole, it is presu ed to contain #ater or fluid #ith a lo# protein content. Li$er" In the liver, ild fatty change ay not affect the gross appearance. 7ith progressive accu ulation, the organ enlarges and beco es increasingly yello# until, in e"tre e instances, the liver ay #eigh < to C 'g and be transfor ed into a bright yello#, soft, greasy organ. )atty change begins #ith the develop ent of inute, e brane&bound inclusions *liposo es. closely applied to the endoplas ic reticulu . )atty change is first seen by light icroscopy as s all vacuoles in the cytoplas around the nucleus. As the process progresses, the vacuoles coalesce, creating cleared spaces that displace the nucleus to the periphery of the cell *)ig. 1&<C .. :ccasionally, contiguous cells rupture, and the enclosed fat globules coalesce, producing so&called fatty cysts. Heart" Kipid is found in cardiac uscle in the for of s all droplets, occurring in t#o patterns. In one, prolonged oderate hypo"ia, such as that produced by profound ane ia, causes intracellular deposits of fat, #hich create grossly apparent bands of yello#ed yocardiu alternating #ith bands of dar'er, red& bro#n, uninvolved yocardiu 1tigered effect2. $he other pattern of hypo"ia is produced by ore profound hypo"ia or by so e for s of yocarditis *e.g., diphtheria. and sho#s ore unifor ly affected yocytes.

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)igure 1&<C )atty liver. A, 9che atic diagra of the possible echanis s leading to accu ulation of triglycerides in fatty liver. Defects in any of the steps of upta'e, catabolis , or secretion can result in lipid accu ulation. , >igh&po#er detail of fatty change of the liver. In ost cells, the #ell&preserved nucleus is s!uee%ed into the displaced ri of cytoplas about the fat vacuole. * , Courtesy of Dr. Ra es Cra#ford, Depart ent of Pathology, 8ale Bniversity 9chool of Medicine, 4e# >aven, C$..

.holesterol a#d .holesterol Esters $he cellular etabolis of cholesterol *discussed in detail in Chapter ,. is tightly regulated such that ost cells use cholesterol for the synthesis of cell e branes #ithout intracellular accu ulation of cholesterol or cholesterol esters. Accu ulations, ho#ever, anifested histologically by intracellular vacuoles, are seen in several pathologic processes. Atherosclerosis. In atherosclerotic pla!ues, s ooth uscle cells and acrophages #ithin the inti al layer of the aorta and large arteries are filled #ith lipid vacuoles, ost of #hich are ade up of cholesterol and cholesterol esters. 9uch cells have a foa y appearance *foa cells., and aggregates of the in the inti a produce the yello# cholesterol&laden athero as characteristic of this serious disorder. 9o e of these fat&laden cells rupture, releasing lipids into the e"tracellular space. $he echanis s of cholesterol accu ulation in both cell types in atherosclerosis are discussed in detail in Chapter 11. $he e"tracellular cholesterol esters ay crystalli%e in the shape of long needles, producing !uite distinctive clefts in tissue sections. 2anthomas. Intracellular accu ulation of cholesterol #ithin acrophages is also

characteristic of ac!uired and hereditary hyperlipide ic states. Clusters of foa y cells are found in the subepithelial connective tissue of the s'in and in tendons, producing tu orous asses 'no#n as "antho as. )nflammation and necrosis. )oa y acrophages are fre!uently found at sites of cell injury and infla ation, o#ing to phagocytosis of cholesterol fro the e branes of injured cells, including parenchy al cells, leu'ocytes, and erythrocytes. Phospholipids and yelin figures are also found in infla atory foci. 7hen abundant, the cholesterol&laden acrophages i part a yello#ish discoloration to such infla atory foci. (holesterolosis. $his refers to the focal accu ulations of cholesterol&laden acrophages in the la ina propria of the gallbladder *)ig. 1&<A.. $he echanis of accu ulation is un'no#n. 3iemann&Pic" disease, type (. In this lysoso al storage disease, an en%y e involved in cholesterol traffic'ing is utated, and hence cholesterol accu ulates in ultiple organs *Chapter ,..

PROTEI,S Intracellular accu ulations of proteins usually appear as rounded, eosinophilic droplets, vacuoles, or aggregates in the cytoplas . By electron icroscopy, they can be a orphous, fibrillar, or crystalline in appearance. In so e disorders, such as certain for s of a yloidosis, abnor al proteins deposit pri arily in the e"tracellular space *Chapter C..

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)igure 1&<A Cholesterolosis. Cholesterol&laden acrophages *foa cells. fro a focus of gallbladder cholesterolosis *arrow.. *Courtesy of Dr. Matthe# 8eh, Bniversity of 7ashington, 9eattle, 7A..

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)igure 1&<D Protein reabsorption droplets in the renal tubular epitheliu . *Courtesy of Dr. >el ut 6enn'e, Depart ent of Pathology, Brigha and 7o en/s >ospital, Boston, MA..

3"cesses of proteins #ithin the cells sufficient to cause orphologically visible accu ulation have diverse causes. 4ea#sorption droplets in proximal renal tu#ules are seen in renal diseases associated #ith protein loss in the urine *proteinuria.. In the 'idney, s all a ounts of protein filtered through the glo erulus are nor ally reabsorbed by pinocytosis in the pro"i al tubule. In disorders #ith heavy protein lea'age across the glo erular filter, there is increased reabsorption of the protein into vesicles. $hese vesicles fuse #ith lysoso es to produce phagolysoso es, #hich appear as pin' hyaline droplets #ithin the cytoplas of the tubular cell *)ig. 1&<D.. $he process is reversible; if the proteinuria di inishes, the protein droplets are etaboli%ed and disappear. A second cause is synthesis of e"cessive a ounts of nor al secretory protein, as occurs in certain plas a cells engaged in active synthesis of i unoglobulins. $he 36 beco es hugely distended, producing large, ho ogeneous eosinophilic inclusions called 4ussell #odies. /efects in protein folding ay underlie so e of these depositions in a variety of unrelated diseases.AC 4ascent polypeptide chains of proteins, ade on riboso es, are ulti ately

arranged into either H helices or E sheets, and the proper configuration of these arrange ents *protein folding. is critical to the individual protein/s function and its transport into cell organelles.AA In the process of folding, partially folded inter ediates arise, and these ay for intracellular aggregates a ong the selves or by entangling other proteins. Bnder nor al conditions, ho#ever, these inter ediates are stabili%ed by a nu ber of olecular chaperones, #hich interact #ith proteins directly. AD Chaperones aid in proper folding and in transport across the 36, 1olgi co ple", and beyond *)ig. 1&<I.. 9o e chaperones are synthesi%ed constitutively and affect nor al intracellular protein traffic'ing, #hereas others are induced by stress, such as heat *heat&shoc' proteins, e.g., hspA-, hspI-., and (rescue( shoc'&stressed proteins fro isfolding. If the folding process is not successful, the chaperones facilitate degradation of the da aged protein. $his degradative process often involves ubi!uitin *also a heat&shoc' protein., #hich is added to the abnor al protein and ar's it for degradation by the proteaso e co ple". $here are several echanis s by #hich protein folding defects can cause intracellular accu ulations or result in disease. o /efective intracellular transport and secretion of critical proteins. In H5&antitrypsin deficiency, utations in the protein significantly slo# folding, resulting in the build& up of partially folded inter ediates, #hich aggregate in the 36 of the liver and are not secreted. $he resultant deficiency of the circulating en%y e causes e physe a *Chapter 1,.. In cystic fi#rosis, utation delays dissociation of a chloride channel protein fro one of its chaperones, resulting in abnor al folding and loss of function *Chapter 1-.. In familial hypercholesterolemia, utations in lo#&density lipoprotein receptors interfere #ith proper folding of receptor proteins *Chapter ,.. o 14 stress induced #y unfolded and misfolded proteins. Bnfolded or isfolded proteins accu ulate in the 36 and trigger a nu ber of cellular responses, collectively called the unfolded protein response.AI&D1 $he unfolded protein response is ediated by several proteins that reside in and span the 36 e brane. $he lu inal do ains of these proteins sense perturbations in protein folding, and the cytoplas ic do ains activate signaling path#ays that reduce the levels of isfolded proteins in the cell, by increasing the production of chaperones and slo#ing do#n protein translation. Parado"ically, the activation of the unfolded protein response also leads to cell death by activating caspases, particularly an 36&resident caspase called caspase&1+. $hus, isfolded proteins initially trigger the cytoprotective function of this response, but if these abnor al proteins persist, the pro&apoptotic cytoto"ic functions ta'e over. Aggregation of abnor ally folded proteins, caused by genetic utations, aging, or un'no#n environ ental factors, is no# recogni%ed as a feature of a nu ber of neurodegenerative diseases, including Al%hei er/s, >untington/s, and Par'inson/s diseases *Chapter +D., and possibly type II diabetes. Deprivation of glucose and o"ygen, and stress such as heat, also result in protein isfolding and trigger the unfolded protein response, cul inating in cell injury and death. o Aggregation of a#normal proteins. Abnor al or isfolded proteins ay deposit in tissues and interfere #ith nor al functions. $he deposits can be intracellular, e"tracellular, or both, and there is accu ulating evidence that the aggregates ay either directly or indirectly cause the pathologic changes. Certain for s of amyloidosis *Chapter C. fall in this category of diseases. $hese disorders are so eti es called proteinopathies or protein&aggregation diseases.

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)igure 1&<I Mechanis s of protein folding and the role of chaperones. A, Chaperones, such as heat shoc' proteins *>sp., protect unfolded or partially folded protein fro degradation and guide proteins into organelles. , Chaperones repair isfolded proteins; #hen this process is ineffective, proteins are targeted for degradation in the proteaso e, and if isfolded proteins accu ulate they trigger apoptosis.

HYALI,E .HA,/E $he ter hyaline usually refers to an alteration #ithin cells or in the e"tracellular space, #hich gives a ho ogeneous, glassy, pin' appearance in routine histologic sections stained #ith he ato"ylin and eosin. It is #idely used as a descriptive histologic ter rather than a specific ar'er for cell injury. $his tinctorial change is produced by a variety of alterations and does not represent a specific pattern of accu ulation. Intracellular accu ulations of protein, described earlier *reabsorption droplets, 6ussell bodies, Mallory alcoholic hyalin., are e"a ples of intracellular hyaline deposits. 1xtracellular hyalin has been so e#hat ore difficult to analy%e. Collagenous fibrous tissue in old scars ay appear hyalini%ed, but the physioche ical echanis underlying this change is not clear. In long&standing hypertension and diabetes ellitus, the #alls of arterioles, especially in the 'idney, beco e hyalini%ed, o#ing to e"travasated plas a protein and deposition of base ent e brane aterial. /LY.O/E, 1lycogen is a readily available energy store that is present in the cytoplas . 3"cessive intracellular deposits of glycogen are seen in patients #ith an abnor ality in either glucose or glycogen etabolis . 7hatever the clinical setting, the glycogen asses appear as clear vacuoles #ithin the cytoplas . 1lycogen is best preserved in nona!ueous fi"atives; for its locali%ation, tissues are best fi"ed in absolute alcohol. 9taining #ith Best car ine or the periodic acid schiff *PA9. reaction i parts a rose&to&violet color to the glycogen, and diastase digestion of a parallel section before staining serves as a further control by hydroly%ing the glycogen. Diabetes ellitus is the pri e e"a ple of a disorder of glucose etabolis . In this disease, glycogen is found in the epithelial cells of the distal portions of the pro"i al convoluted tubules and so eti es in the descending loop of >enle, as #ell as #ithin liver cells, E cells of the islets of Kangerhans, and heart uscle cells. 1lycogen also accu ulates #ithin the cells in a group of closely related disorders, all genetic, collectively referred to as the glycogen storage diseases, or glycogenoses *Chapter ,.. In these diseases, en%y atic defects in the synthesis or brea'do#n of glycogen result in assive accu ulation, #ith secondary injury and cell death. PI/*E,TS Pig ents are colored substances, so e of #hich are nor al constituents of cells *e.g., elanin., #hereas others are abnor al and collect in cells only under special circu stances. Pig ents can be e"ogenous, co ing fro outside the body, or endogenous, synthesi%ed #ithin the body itself. E*ogenous Pigments. $he ost co on exogenous pigment is car#on or coal dust, #hich is a ubi!uitous air pollutant of urban life. 7hen inhaled, it is pic'ed up by acrophages #ithin the alveoli and is then transported through ly phatic channels to the regional ly ph nodes in the tracheobronchial region. Accu ulations of this pig ent blac'en the tissues of the lungs (anthracosis) and the involved ly ph nodes. In coal iners, the aggregates of carbon dust ay induce a fibroblastic reaction or even e physe a and thus cause a serious lung disease 'no#n as coal wor"er6s pneumoconiosis *Chapter 1,.. +attooing is a for of locali%ed, e"ogenous

pig entation of the s'in. $he pig ents inoculated are phagocytosed by der al acrophages, in #hich they reside for the re ainder of the life of the e bellished *so eti es #ith e barrassing conse!uences for the bearer of the tattooU.. $he pig ents do not usually evo'e any infla atory response. Endogenous Pigments. $ipofuscin is an insoluble pig ent, also 'no#n as lipochro e and #ear& and&tear or aging pig ent. Kipofuscin is co posed of poly ers of lipids and phospholipids co ple"ed #ith protein, suggesting that it is derived through lipid pero"idation of polyunsaturated lipids of subcellular e branes. Kipofuscin is not injurious to the cell or its functions. Its i portance lies in its being the telltale sign of free radical injury and lipid pero"idation. $he ter is derived fro the Katin *fuscus V bro#n., thus bro#n lipid. In tissue sections, it appears as a yello#& bro#n, finely granular intracytoplas ic, often perinuclear pig ent *)ig. 1&@-.. It is seen in cells undergoing slo#, regressive changes and is particularly pro inent in the liver and heart of aging patients or patients #ith severe alnutrition and cancer cache"ia. :n electron icroscopy, the granules are highly electron dense, often have e branous structures in their idst, and are usually in a perinuclear location. 'elanin, derived fro the 1ree' *melas V blac'., is an endogenous, non&he oglobin&derived, bro#n&blac' pig ent for ed #hen the en%y e tyrosinase cataly%es the o"idation of tyrosine to dihydro"yphenylalanine in elanocytes. It is discussed further in Chapter +,. )or all practical purposes, elanin is the only endogenous #rown&#lac" pigment. $he only other that could be considered in this category is ho ogentisic acid, a blac' pig ent that occurs in patients #ith al"aptonuria, a rare etabolic disease. >ere the pig ent is deposited in the s'in, connective tissue, and cartilage, and the pig entation is 'no#n as ochronosis *Chapter ,.. Hemosiderin is a he oglobin&derived, golden yello#&to&bro#n, granular or crystalline pig ent in #hich for iron is stored in cells. Iron etabolis and the synthesis of ferritin and he osiderin are considered in detail in Chapter 1<. Iron is nor ally carried by specific transport proteins, transferrins. In cells, it is stored in association #ith a protein, apoferritin, to for ferritin icelles. )erritin is a constituent of ost cell types. *hen there is a local or systemic excess of iron, ferritin forms hemosiderin granules, #hich are easily seen #ith the light icroscope *)ig. 1&@1.. $hus, he osiderin pig ent represents aggregates of ferritin icelles. Bnder nor al conditions, s all a ounts of he osiderin can be seen in the ononuclear phagocytes of the bone arro#, spleen, and liver, all actively engaged in red cell brea'do#n.
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)igure 1&@- Kipofuscin granules in a cardiac yocyte as sho#n by A, light icroscopy *deposits indicated by arrows., and , electron icroscopy *note the perinuclear, intralysoso al location..

3"cesses of iron cause he osiderin to accu ulate #ithin cells, either as a locali%ed process or as a syste ic derange ent. $ocal excesses of iron and he osiderin result fro gross he orrhages or the yriad inute he orrhages that acco pany severe vascular congestion. $he best e"a ple of locali%ed he osiderosis is the co on bruise. After local he orrhage, the area is at first red& blue. 7ith lysis of the erythrocytes, the he oglobin eventually undergoes transfor ation to he osiderin. Macrophages ta'e part in this process by phagocytiosing the red cell debris, and then lysoso al en%y es eventually convert the he oglobin, through a se!uence of pig ents, into he osiderin. $he play of colors through #hich the bruise passes reflects these transfor ations. $he original red&blue color of he oglobin is transfor ed to varying shades of green&blue, co prising the local for ation of biliverdin *green bile., then bilirubin *red bile., and thereafter the iron oiety of he oglobin is deposited as golden yello# he osiderin. 7henever there are causes for systemic overload of iron, he osiderin is deposited in any

organs and tissues, a condition called hemosiderosis. It is seen #ith0 *1. increased absorption of dietary iron, *+. i paired use of iron, *<. he olytic ane ias, and *@. transfusions because the transfused red cells constitute an e"ogenous load of iron. $hese conditions are discussed in Chapter 1D.

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Morphology. Iron pig ent appears as a coarse, golden, granular pig ent lying #ithin the cell/s cytoplas . 7hen the basic cause is the locali%ed brea'do#n of red cells, the pig entation is found at first in the phagocytes in the area. In syste ic he osiderosis, it is found at first in the ononuclear phagocytes of the liver, bone arro#, spleen, and ly ph nodes and in scattered acrophages throughout other organs such as the s'in, pancreas, and 'idneys. 7ith progressive accu ulation, parenchy al cells throughout the body *principally in the liver, pancreas, heart, and endocrine organs. beco e pig ented. Iron can be visuali%ed in tissues by the Prussian blue histoche ical reaction, in #hich colorless potassiu ferrocyanide is converted by iron to blue&blac' ferric ferrocyanide *)ig. 1&@1 ..
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In ost instances of syste ic he osiderosis, the pig ent does not da age the parenchy al cells or i pair organ function. $he ore e"tre e accu ulation of iron, ho#ever, in a disease called hemochromatosis, is associated #ith liver, heart, and pancreatic da age, resulting in liver fibrosis, heart failure, and diabetes ellitus *Chapter 1D..
)igure 1&@1 >e osiderin granules in liver cells. A, >S3 section sho#ing golden&bro#n, finely granular pig ent. , Prussian blue reaction, specific for iron.

iliru#in is the nor al ajor pig ent found in bile. It is derived fro he oglobin but contains no iron. Its nor al for ation and e"cretion are vital to health, and jaundice is a co on clinical disorder caused by e"cesses of this pig ent #ithin cells and tissues. Bilirubin etabolis and jaundice are discussed in Chapter 1D.

Pathologic Calcification
Pathologic calcification is the abnor al tissue deposition of calciu salts, together #ith s aller a ounts of iron, agnesiu , and other ineral salts. It is a co on process occurring in a variety of pathologic conditions. $here are t#o for s of pathologic calcification. 7hen the deposition occurs locally in dying tissues, it is 'no#n as dystrophic calcification; it occurs despite nor al seru levels of calciu and in the absence of derange ents in calciu etabolis . In contrast, the deposition of calciu salts in other#ise nor al tissues is 'no#n as metastatic calcification, and it al ost al#ays results fro hypercalce ia secondary to so e disturbance in calciu etabolis . DYSTROPHI. .AL.I-I.ATIO, Dystrophic calcification is encountered in areas of necrosis, #hether they are of coagulative, caseous, or li!uefactive type, and in foci of en%y atic necrosis of fat. Calcification is al ost inevitable in the athero as of advanced atherosclerosis. It also co only develops in aging or da aged heart valves, further ha pering their function *)ig. 1&@+.. 7hatever the site of deposition, the calciu salts appear acroscopically as fine, #hite granules or clu ps, often felt as gritty deposits. 9o eti es a tuberculous ly ph node is virtually converted to stone.

Morphology. >istologically, #ith the usual he ato"ylin and eosin stain, the calciu salts have a basophilic, a orphous granular, so eti es clu ped, appearance. $hey can be intracellular, e"tracellular, or in both locations. In the course of ti e, heterotopic bone ay be for ed in the focus of calcification. :n occasion, single necrotic cells ay constitute seed crystals that beco e encrusted by the ineral deposits. $he progressive ac!uisition of outer layers ay create la ellated configurations, called psammoma &odies because of their rese blance to grains of sand. 9o e types of papillary cancers *e.g., thyroid. are apt to develop psa o a bodies. 9trange concretions e erge #hen calciu iron salts gather about long slender spicules of asbestos in the lung, creating e"otic, beaded du bbell for s.

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)igure 1&@+ 5ie# loo'ing do#n onto the unopened aortic valve in a heart #ith calcific aortic stenosis. $he se ilunar cusps are thic'ened and fibrotic. Behind each cusp are seen irregular asses of piled&up dystrophic calcification.

Pathogenesis. In the pathogenesis of dystrophic calcification, the final co on path#ay is the for ation of crystalline calciu phosphate ineral in the for of an apatite si ilar to the hydro"yapatite of bone. $he process has t#o ajor phases0 initiation *or nucleation. and propagation; both can occur intracellularly and e"tracellularly. Initiation of intracellular calcification occurs in the mitochondria of dead or dying cells that accu ulate calciu . Initiators of extracellular dystrophic calcification include phospholipids found in e brane&bound vesicles about +-- n in dia eter; in cartilage and bone, they are 'no#n as matrix vesicles, and in pathologic calcification, they are derived fro degenerating or aging cells. It is thought that calciu is concentrated in these vesicles by a process of e brane&facilitated calcification, #hich has several steps0 *1. calciu ion binds to the phospholipids present in the vesicle e brane, *+. phosphatases associated #ith the e brane generate phosphate groups, #hich bind to the calciu , *<. the cycle of calciu and phosphate binding is repeated, raising the local concentrations and producing a deposit near the e brane, and *@. a structural change occurs in the arrange ent of calciu and phosphate groups, generating a icrocrystal, #hich can then propagate and perforate the e brane. Propagation of crystal for ation depends on the concentration of Ca +N and P:@ and the presence of inhibitors and other proteins in the e"tracellular space, such as the connective tissue atri" proteins. Although dystrophic calcification ay be si ply a telltale sign of previous cell injury, it is often a cause of organ dysfunction. 9uch is the case in calcific valvular disease and atherosclerosis, as beco es clear in further discussion of these diseases. *ETASTATI. .AL.I-I.ATIO,
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Metastatic calcification ay occur in nor al tissues #henever there is hypercalce ia. >ypercalce ia also accentuates dystrophic calcification. $here are four principal causes of hypercalce ia0 *1. increased secretion of parathyroid hor one *P$>. #ith subse!uent bone resorption, as in hyperparathyroidis due to parathyroid tu ors, and ectopic secretion of P$>& related protein by alignant tu ors *Chapter A.; *+. destruction of #one tissue, occurring #ith pri ary tu ors of bone arro# *e.g., ultiple yelo a, leu'e ia. or diffuse s'eletal etastasis *e.g., breast cancer., accelerated bone turnover *e.g., Paget disease., or i obili%ation; *<. vitamin /&related disorders, including vita in D into"ication, sarcoidosis *in #hich acrophages activate a vita in D precursor., and idiopathic hypercalce ia of infancy *7illia s syndro e., characteri%ed by abnor al sensitivity to vita in D; and *@. renal failure, #hich causes retention of phosphate, leading to secondary hyperparathyroidis . Kess co on causes include alu inu into"ication, #hich occurs in patients on chronic renal dialysis, and il'&al'ali syndro e, #hich is

due to e"cessive ingestion of calciu .

and absorbable antacids such as

il' or calciu

carbonate

Metastatic calcification ay occur #idely throughout the body but principally affects the interstitial tissues of the gastric ucosa, 'idneys, lungs, syste ic arteries, and pul onary veins. Although !uite different in location, all of these tissues lose acid and therefore have an internal al'aline co part ent that predisposes the to etastatic calcification. In all these sites, the calciu salts orphologically rese ble those described in dystrophic calcification. $hus, they ay occur as noncrystalline a orphous deposits or, at other ti es, as hydro"yapatite crystals. Bsually, the ineral salts cause no clinical dysfunction, but, on occasion, assive involve ent of the lungs produces re ar'able "&ray fil s and respiratory deficits. Massive deposits in the 'idney *nephrocalcinosis. ay in ti e cause renal da age *Chapter +-..

Cellular

ging

9ha'espeare probably characteri%ed aging best in his elegant description of the seven ages of an. It begins at the o ent of conception, involves the differentiation and aturation of the organis and its cells, at so e variable point in ti e leads to the progressive loss of functional capacity characteristic of senescence, and ends in death. 7ith age, there are physiologic and structural alterations in al ost all organ syste s. Aging in individuals is affected to a great e"tent by genetic factors, diet, social conditions, and occurrence of age&related diseases, such as atherosclerosis, diabetes, and osteoarthritis. In addition, there is good evidence that aging&induced alterations in cells are an i portant co ponent of the aging of the organis . >ere #e discuss cellular aging because it could represent the progressive accu ulation over the years of sublethal injury that ay lead to cell death or at least to the di inished capacity of the cell to respond to injury. (ellular aging is the result of a progressive decline in the proliferative capacity and life span of cells and the effects of continuous exposure to exogenous influences that result in the progressive accumulation of cellular and molecular damage *)ig. 1&@<.. $hese processes are revie#ed ne"t. $tructural and 5iochemical Changes !ith Cellular ging. A nu ber of cell functions decline progressively #ith age. :"idative phosphorylation by itochondria is reduced, as is synthesis of nucleic acids and structural and en%y atic proteins, cell receptors, and transcription factors. 9enescent cells have a decreased capacity for upta'e of nutrients and for repair of chro oso al da age. $he orphologic alterations in aging cells include irregular and abnor ally lobed nuclei, pleo orphic vacuolated itochondria, decreased endoplas ic reticulu , and distorted 1olgi apparatus. Conco itantly, there is a steady accu ulation of the pig ent lipofuscin, #hich, as #e have seen, represents a product of lipid pero"idation and evidence of oxidative damage7 advanced glycation end products, #hich result fro nonen%y atic glycosylation and are capable of cross& lin'ing adjacent proteins; and the accu ulation of a#normally folded proteins. $he role of o"idative da age is discussed later. Advanced glycation end products are i portant in the pathogenesis of diabetes ellitus and are discussed in Chapter +@, but they ay also participate in aging. )or e"a ple, age&related glycosylation of lens proteins ay underlie senile cataracts. $he nature of abnor ally folded proteins #as discussed earlier in the chapter.

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)igure 1&@< Mechanis s of cellular aging. 1enetic factors and environ ental insults co bine to produce the cellular abnor alities characteristic of aging.

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)igure 1&@@ )inite population doublings of pri ary hu an fibroblasts derived fro a ne#born, a 1--&year&old person, and a +-&year&old patient #ith 7erner/s syndro e. $he ability of cells to gro# to a confluent onolayer decreases #ith increasing population&doubling levels. *)ro Dice R)0 Cellular and olecular echanis s of aging. Physiol 6ev A<01,-, 1II<..

'eplicati(e $enescence. $he concept that cells have a li ited capacity for replication #as developed fro a si ple e"peri ental odel for aging. 4or al hu an fibroblasts, #hen placed in tissue culture, have li ited division potential.D+ Cells fro children undergo ore rounds of replication than cells fro older people *)ig. 1&@@.. In contrast, cells fro patients #ith *erner syndrome, a rare disease characteri%ed by pre ature aging, have a ar'edly reduced in vitro life span. After a fi"ed nu ber of divisions, all cells beco e arrested in a ter inally nondividing state, 'no#n as cellular senescence. Many changes in gene e"pression occur during cellular aging, but a 'ey !uestion is #hich of these are causes and #hich are effects of cellular senescence. D< )or e"a ple, so e of the proteins that inhibit progression of the cell gro#th cycle *as detailed in Chapter A.&such as the products of the cyclin&dependent 'inase inhibitor genes *e.g., p85.&are overe"pressed in senescent cells. >o# dividing cells can count their divisions is under intensive investigation. :ne li'ely echanis is that #ith each cell division, there is incomplete replication of chromosome ends (telomere shortening), which ultimately results in cell cycle arrest . +elomeres are short repeated se!uences of D4A *$$A111. present at the linear ends of chro oso es that are i portant for ensuring the co plete replication of chro oso e ends and protecting chro oso al ter ini fro fusion and degradation.D@,D, 7hen so atic cells replicate, a s all section of the telo ere is not duplicated, and telo eres beco e progressively shortened. As the telo eres beco e shorter, the ends of chro oso es cannot be protected and are seen as bro'en D4A, #hich signals cell cycle arrest. $he lengths of the telo eres are nor ally aintained by nucleotide addition ediated by an en%y e called telomerase. $elo erase is a speciali%ed 64A&protein co ple" that uses its o#n 64A as a te plate for adding nucleotides to the ends of chro oso es *)ig. 1&@,.. $he activity of telo erase is repressed by regulatory proteins, #hich restrict telo ere elongation, thus providing a length sensing echanis . $elo erase activity is e"pressed in ger cells and is present at lo# levels in ste cells, but it is usually absent in ost so atic tissues. $herefore, as cells age, their telo eres beco e shorter, and they e"it the cell cycle, resulting in an inability to generate ne# cells to replace da aged ones. Conversely, in i ortal cancer cells, telo erase is reactivated, and telo eres are not shortened, suggesting that telo ere elongation ight be an i portant&possibly essential&step in tu or for ation.D, Despite such alluring observations, ho#ever, the relationship of telo erase activity and telo eric length to aging and cancer still needs to be fully established. DC Genes That Influence the ging Process. 9tudies in /rosophila, (. elegans, and ice are leading to the discovery of genes that influence the aging process. DA :ne interesting set of genes involves the insulinPinsulin gro#th factor&1 path#ay. Decreased signaling through the I1)&1 receptor as a result of decreased caloric inta'e, or utations in the receptor, result in prolonged life span in (. elegans. $he signals do#nstrea of the I1)&1 receptor involve a nu ber of 'inases and ay lead to the silencing of particular genes, thus pro oting aging. Analyses of hu ans #ith pre ature aging are also establishing the funda ental concept that aging is not a rando process but is regulated by specific genes, receptors, and signals. DD ccumulation of Meta&olic and Genetic Damage. In addition to the i portance of ti ing and a genetic cloc', cellular life span ay also be deter ined by the balance bet#een cellular da age resulting fro meta#olic events occurring #ithin the cell and counteracting olecular responses that can repair the da age. 9 aller ani als have generally shorter life spans and faster etabolic rates, suggesting that the life span of a species is li ited by fi"ed total etabolic consu ption over

a lifeti e.DI :ne group of products of nor al etabolis are reactive o"ygen species. As #e have seen, these byproducts of o"idative phosphorylation cause covalent odifications of proteins, lipids, and nucleic acids. $he a ount of o"idative da age, #hich increases as an organis ages, ay be an i portant co ponent of senescence, and the accu ulation of lipofuscin in aging cells is seen as the telltale sign of such da age. Consistent #ith this proposal are the follo#ing observations0 *1. variation in longevity a ong different species is inversely correlated #ith the rates of itochondrial generation of supero"ide anion radical, and *+. overe"pression of the antio"idative en%y es supero"ide dis utase *9:D. and catalase e"tends life span in transgenic for s of /rosophila. $hus, part of the echanis that ti es aging ay be the cu ulative da age that is generated by to"ic byproducts of etabolis , such as o"ygen radicals. Increased o"idative da age could result fro repeated environ ental e"posure to such influences as ioni%ing radiation, progressive reduction of antio"idant defense echanis s *e.g., vita in 3 , glutathione pero"idase., or both. A nu ber of protective responses counterbalance progressive da age in cells, and an i portant one is the recognition and repair of da aged D4A. I- Although ost D4A da age is repaired by endogenous D4A repair en%y es, so e persists and accu ulates as cells age. 9everal lines of evidence point to the i portance of D4A repair in the aging process. Patients #ith *erner syndrome sho# pre ature aging, and the defective gene product is a D4A helicase & a protein involved in D4A replication and repair and other functions re!uiring D4A un#inding. I1 A defect in this en%y e causes rapid accu ulation of chro oso al da age that i ics the injury that nor ally accu ulates during cellular aging. 1enetic instability in so atic cells is also characteristic of other disorders in #hich patients display so e of the anifestations of aging at an increased rate, such as ataxia&telangiectasia, in #hich the utated gene encodes a protein involved in repairing double strand brea's in D4A *Chapter A.. 9tudies of utants of budding yeast and (. elegans sho# that life span is increased if responses to D4A da age are enhanced. $hus, the balance bet#een cu ulative etabolic da age and the response to that da age could deter ine the rate at #hich #e age. In this scenario, aging can be delayed by decreasing the accu ulation of da age or by increasing the response to that da age.
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)igure 1&@, $he role of telo eres and telo erase in replicative senescence of cells. A, $elo erase directs 64A te plate&dependent D4A synthesis, in #hich nucleotides are added to one strand at the end of a chro oso e. $he lagging strand is presu ably filled in by D4A poly erase H. $he 64A se!uence in the telo erase is different in different species. *Modified fro Alberts B6, et al0 Molecular Biology of the Cell, +--+, 1arland 9cience, 4e# 8or'.. , $elo ere&telo erase hypothesis and proliferative capacity. $elo ere length is plotted against the nu ber of cell divisions. In nor al so atic cells, there is no telo erase activity, and telo eres progressively shorten #ith increasing cell divisions until gro#th arrest, or senescence, occurs. 1er cells and ste cells both contain active telo erase, but only the ger cells have sufficient levels of the en%y e to stabili%e telo ere length co pletely. $elo erase activation in cancer cells inactivates the teleo eric cloc' that li its the proliferative capacity of nor al so atic cells. *Modified and redra#n #ith per ission fro >olt 93, et al.0 6efining the telo er& telo erase hypothesis of aging and cancer. 4ature Biotech 1@0D<C, 1IIC. Copyright 1IIC, Mac illan Maga%ines Ki ited..

4ot only da aged D4A but da aged cellular organelles also accu ulate as cells age. In part, this ay be the result of declining function of the proteaso e, the proteolytic achine that serves to eli inate abnor al and un#anted intracellular proteins. I+ In conclusion, it should be apparent that the various for s of cellular derange ents and

adaptations described in this chapter cover a #ide spectru , ranging fro adaptations in cell si%e, gro#th, and function; to the reversible and irreversible for s of acute cell injury; to the regulated type of cell death represented by apoptosis; to the pathologic alterations in cell organelles; and to the less o inous for s of intracellular accu ulations, including pig entations. 6eference is ade to all these alterations throughout this boo' because all organ injury and ulti ately all clinical disease arise fro derange ents in cell structure and function. 'EFE'ENCE$
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