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(BQ) Part 2 book “Marks’ basic medical biochemistry: A clinical approach’ has contents: Gluconeogenesis and maintenance of blood glucose levels, digestion and transport of dietary lipids, liver metabolism, blood plasma proteins, coagulation and fibrinolysis, the biochemistry of the erythrocyte and other blood cells,… and other contents.
24 Oxygen Toxicity and Free Radical Injury O2 is both essential to human life and toxic We are dependent on O2 for oxidation reactions in the pathways of adenosine triphosphate (ATP) generation, detoxification, and biosynthesis However, when O2 accepts single electrons, it is transformed into highly reactive oxygen radicals that damage cellular lipids, proteins, and DNA Damage by reactive oxygen radicals contributes to cellular death and degeneration in a wide range of diseases (Table 24.1) Radicals are compounds that contain a single electron, usually in an outside orbital Oxygen is a biradical, a molecule that has two unpaired electrons in separate orbitals (Fig 24.1) Through a number of enzymatic and nonenzymatic processes that routinely occur in cells, O2 accepts single electrons to form reactive oxygen species (ROS) ROS are highly reactive oxygen radicals, or compounds that are readily converted in cells to these reactive radicals The ROS formed by reduction of O2 are the radical superoxide (O2¯ ), the nonradical hydrogen peroxide (H2O2 ), and the hydroxyl radical (OH• ) ROS may be generated nonenzymatically, or enzymatically as accidental byproducts or major products of reactions Superoxide may be generated nonenzymatically from CoQ, or from metal-containing enzymes (e.g., cytochrome P450, xanthine oxidase, and NADPH oxidase) The highly toxic hydroxyl radical is formed nonenzymatically from superoxide in the presence of Fe3ϩ or Cuϩ by the Fenton reaction, and from hydrogen peroxide in the Haber–Weiss reaction Oxygen radicals and their derivatives can be deadly to cells The hydroxyl radical causes oxidative damage to proteins and DNA It also forms lipid peroxides and malondialdehyde from membrane lipids containing polyunsaturated fatty acids In some cases, free radical damage is the direct cause of a disease state (e.g., tissue damage initiated by exposure to ionizing radiation) In neurodegenerative diseases, such as Parkinson’s disease, or in ischemia-reperfusion injury, ROS may perpetuate the cellular damage caused by another process Oxygen radicals are joined in their destructive damage by the free radical nitric oxide (NO) and the reactive oxygen species hypochlorous acid (HOCl) NO Oxygen is a biradical O2 which forms – ROS O2 H2O2 OH• Fig 24.1 O2 is a biradical It has two antibonding electrons with parallel spins, denoted by the parallel arrows It has a tendency to form toxic reactive oxygen species (ROS), such as superoxide (O2Ϫ), the nonradical hydrogen peroxide (H2O2), and the hydroxyl radical (OH•) Table 24.1 Some Disease States Associated with Free Radical Injury Atherogenesis Emphysema bronchitis Duchenne-type muscular dystrophy Pregnancy/preeclampsia Cervical cancer Alcohol-induced liver disease Hemodialysis Diabetes Acute renal failure Aging Retrolental fibroplasia Cerebrovascular disorders Ischemia/reperfusion injury Neurodegenerative disorders Amyotrophic lateral sclerosis (Lou Gehrig’s disease) Alzheimer’s disease Down’s syndrome Ischemia/reperfusion injury following stroke Oxphos diseases (Mitochondrial DNA disorders) Multiple sclerosis Parkinson’s disease 439 440 SECTION FOUR / FUEL OXIDATION AND THE GENERATION OF ATP Cell defenses: Antioxidants Enzymes ROS RNOS Oxidative stress Fig 24.2 Oxidative stress Oxidative stress occurs when the rate of ROS and RNOS production overbalances the rate of their removal by cellular defense mechanisms These defense mechanisms include a number of enzymes and antioxidants Antioxidants usually react nonenzymatically with ROS The basal ganglia are part of a neuronal feedback loop that modulates and integrates the flow of information from the cerebral cortex to the motor neurons of the spinal cord The neostriatum is the major input structure from the cerebral cortex The substantia nigra pars compacta consists of neurons that provide integrative input to the neostriatum through pigmented neurons that use dopamine as a neurotransmitter (the nigrastriatal pathway) Integrated information feeds back to the basal ganglia and to the cerebral cortex to control voluntary movement In Parkinson’s disease, a decrease in the amount of dopamine reaching the basal ganglia results in the movement disorder In ventricular fibrillation, rapid premature beats from an irritative focus in ventricular muscle occur in runs of varying duration Persistent fibrillation compromises cardiac output, leading to death This arrythmia can result from severe ischemia (lack of blood flow) in the ventricular muscle of the heart caused by clots forming at the site of a ruptured atherosclerotic plaque However, Cora Nari’s rapid beats began during the infusion of TPA as the clot was lysed Thus, they probably resulted from reperfusing a previously ischemic area of her heart with oxygenated blood This phenomenon is known as ischemia–reperfusion injury, and it is caused by cytotoxic ROS derived from oxygen in the blood that reperfuses previously hypoxic cells Ischemic–reperfusion injury also may occur when tissue oxygenation is interrupted during surgery or transplantation combines with O2 or superoxide to form reactive nitrogen oxygen species (RNOS), such as the nonradical peroxynitrite or the radical nitrogen dioxide RNOS are present in the environment (e.g., cigarette smoke) and generated in cells During phagocytosis of invading microorganisms, cells of the immune system produce O2¯ , HOCl, and NO through the actions of NADPH oxidase, myeloperoxidase, and inducible nitric oxide synthase, respectively In addition to killing phagocytosed invading microorganisms, these toxic metabolites may damage surrounding tissue components Cells protect themselves against damage by ROS and other radicals through repair processes, compartmentalization of free radical production, defense enzymes, and endogenous and exogenous antioxidants (free radical scavengers) The defense enzyme superoxide dismutase (SOD) removes the superoxide free radical Catalase and glutathione peroxidase remove hydrogen peroxide and lipid peroxides Vitamin E, vitamin C, and plant flavonoids act as antioxidants Oxidative stress occurs when the rate of ROS generation exceeds the capacity of the cell for their removal (Fig 24.2) THE WAITING ROOM Two years ago, Les Dopaman (less dopamine), a 62-year-old man, noted an increasing tremor of his right hand when sitting quietly (resting tremor) The tremor disappeared if he actively used this hand to purposeful movement As this symptom progressed, he also complained of stiffness in his muscles that slowed his movements (bradykinesia) His wife noticed a change in his gait; he had begun taking short, shuffling steps and leaned forward as he walked (postural imbalance) He often appeared to be staring ahead with a rather immobile facial expression She noted a tremor of his eyelids when he was asleep and, recently, a tremor of his legs when he was at rest Because of these progressive symptoms and some subtle personality changes (anxiety and emotional lability), she convinced Les to see their family doctor The doctor suspected that her patient probably had primary or idiopathic parkinsonism (Parkinson’s disease) and referred Mr Dopaman to a neurologist In Parkinson’s disease, neurons of the substantia nigra pars compacta, containing the pigment melanin and the neurotransmitter dopamine, degenerate Cora Nari had done well since the successful lysis of blood clots in her coronary arteries with the use of intravenous recombinant tissue plasminogen activator (TPA)(see Chapters 19 and 21) This therapy had quickly relieved the crushing chest pain (angina) she experienced when she won the lottery At her first office visit after discharge from the hospital, Cora’s cardiologist told her she had developed multiple premature contractions of the ventricular muscle of her heart as the clots were being lysed This process could have led to a life-threatening arrhythmia known as ventricular fibrillation However, Cora’s arrhythmia responded quickly to pharmacologic suppression and did not recur during the remainder of her hospitalization I O2 AND THE GENERATION OF ROS The generation of reactive oxygen species from O2 in our cells is a natural everyday occurrence They are formed as accidental products of nonenzymatic and enzymatic CHAPTER 24 / OXYGEN TOXICITY AND FREE RADICAL INJURY reactions Occasionally, they are deliberately synthesized in enzyme-catalyzed reactions Ultraviolet radiation and pollutants in the air can increase formation of toxic oxygen-containing compounds A The Radical Nature of O2 A radical, by definition, is a molecule that has a single unpaired electron in an orbital A free radical is a radical capable of independent existence (Radicals formed in an enzyme active site during a reaction, for example, are not considered free radicals unless they can dissociate from the protein to interact with other molecules.) Radicals are highly reactive and initiate chain reactions by extracting an electron from a neighboring molecule to complete their own orbitals Although the transition metals (e.g., Fe, Cu, and Mo) have single electrons in orbitals, they are not usually considered free radicals because they are relatively stable, not initiate chain reactions, and are bound to proteins in the cell The oxygen atom is a biradical, which means it has two single electrons in different orbitals These electrons cannot both travel in the same orbital because they have parallel spins (spin in the same direction) Although oxygen is very reactive from a thermodynamic standpoint, its single electrons cannot react rapidly with the paired electrons found in the covalent bonds of organic molecules As a consequence, O2 reacts slowly through the acceptance of single electrons in reactions that require a catalyst (such as a metal-containing enzyme) O2 is capable of accepting a total of four electrons, which reduces it to water (Fig 24.3) When O2 accepts one electron, superoxide is formed Superoxide is still a radical because it has one unpaired electron remaining This reaction is not thermodynamically favorable and requires a moderately strong reducing agent that can donate single electrons (e.g., CoQH· in the electron transport chain) When superoxide accepts an electron, it is reduced to hydrogen peroxide, which is not a radical The hydroxyl radical is formed in the next one-electron reduction step in the reduction sequence Finally, acceptance of the last electron reduces the hydroxyl radical to H2O 441 The two unpaired electrons in oxygen have the same (parallel) spin and are called antibonding electrons In contrast, carbon–carbon and carbon–hydrogen bonds each contain two electrons, which have antiparallel spins and form a thermodynamically stable pair As a consequence, O2 cannot readily oxidize a covalent bond because one of its electrons would have to flip its spin around to make new pairs The difficulty in changing spins is called the spin restriction Without the spin restriction, organic life forms could not have developed in the oxygen atmosphere on earth because they would be spontaneously oxidized by O2 Instead, O2 is confined to slower one-electron reactions catalyzed by metals (or metalloenzymes) O2 Oxygen e– – O2 Superoxide e–, 2H+ H2O2 Hydrogen peroxide e–, H+ B Characteristics of Reactive Oxygen Species Reactive oxygen species (ROS) are oxygen-containing compounds that are highly reactive free radicals, or compounds readily converted to these oxygen free radicals in the cell The major oxygen metabolites produced by one-electron reduction of oxygen (superoxide, hydrogen peroxide, and the hydroxyl radical) are classified as ROS (Table 24.2) Reactive free radicals extract electrons (usually as hydrogen atoms) from other compounds to complete their own orbitals, thereby initiating free radical chain reactions The hydroxyl radical is probably the most potent of the ROS It initiates chain reactions that form lipid peroxides and organic radicals and adds directly to compounds The superoxide anion is also highly reactive, but has limited lipid solubility and cannot diffuse far However, it can generate the more reactive hydroxyl and hydroperoxy radicals by reacting nonenzymatically with hydrogen peroxide in the Haber–Weiss reaction (Fig 24.4) Hydrogen peroxide, although not actually a radical, is a weak oxidizing agent that is classified as an ROS because it can generate the hydroxyl radical (OH•) Transition metals, such as Fe2ϩ or Cuϩ, catalyze formation of the hydroxyl radical from hydrogen peroxide in the nonenzymatic Fenton reaction (see Fig 24.4.) H2O + OH • Hydroxyl radical e–, H+ H2O Fig 24.3 Reduction of oxygen by four oneelectron steps The four one-electron reduction steps for O2 progressively generate superoxide, hydrogen peroxide, and the hydroxyl radical plus water Superoxide is sometimes written O2¯· to better illustrate its single unpaired electron H2O2, the half-reduced form of O2, has accepted two electrons and is, therefore, not an oxygen radical To decrease occurrence of the Fenton reaction, accessibility to transition metals, such as Fe2ϩ and Cuϩ , are highly restricted in cells, or in the body as a whole Events that release iron from cellular storage sites, such as a crushing injury, are associated with increased free radical injury 442 SECTION FOUR / FUEL OXIDATION AND THE GENERATION OF ATP Table 24.2 Reactive Oxygen Species (ROS) and Reactive Nitrogen–Oxygen Species (RNOS) Reactive Species Properties O2Ϫ Superoxide anion Produced by the electron transport chain and at other sites Cannot diffuse far from the site of origin Generates other ROS H2O2 Hydrogen peroxide Not a free radical, but can generate free radicals by reaction with a transition metal (e.g., Fe2ϩ ) Can diffuse into and through cell membranes OH• Hydroxyl radical The most reactive species in attacking biologic molecules Produced from H2O2 in the Fenton reaction in the presence of Fe2ϩ or Cuϩ RO•·, R•, R-S• Organic radicals Organic free radicals (R denotes remainder of the compound.) Produced from ROH, RH (e.g., at the carbon of a double bond in a fatty acid) or RSH by OH•· attack RCOO•· Peroxyl radical An organic peroxyl radical, such as occurs during lipid degradation (also denoted LOO•) HOCl Hypochlorous acid Produced in neutrophils during the respiratory burst to destroy invading organisms Toxicity is through halogenation and oxidation reactions Attacking species is OClϪ O2 Tc Singlet oxygen Oxygen with antiparallel spins Produced at high oxygen tensions from absorption of uv light Decays so fast that it is probably not a significant in vivo source of toxicity NO Nitric oxide RNOS A free radical produced endogenously by nitric oxide synthase Binds to metal ions Combines with O2 or other oxygen-containing radicals to produce additional RNOS ONOOϪ Peroxynitrite RNOS A strong oxidizing agent that is not a free radical It can generate NO2 (nitrogen dioxide), which is a radical The Haber–Weiss reaction – + O2 H2O2 Superoxide Hydrogen peroxide H+ O2 + + H2O Oxygen Water •OH Hydroxyl radical The Fenton reaction H2O2 Hydrogen peroxide Fe2+ Fe3+ •OH Hydroxyl radical + OH– Hydroxyl ion Fig 24.4 Generation of the hydroxyl radical by the nonenzymatic Haber–Weiss and Fenton reactions In the simplified versions of these reactions shown here, the transfer of single electrons generates the hydroxyl radical ROS are shown in blue In addition to Fe2ϩ, Cuϩ and many other metals can also serve as singleelectron donors in the Fenton reaction Because hydrogen peroxide is lipid soluble, it can diffuse through membranes and generate OH• at localized Fe2ϩ- or Cuϩ-containing sites, such as the mitochondria Hydrogen peroxide is also the precursor of hypochlorous acid (HOCl), a powerful oxidizing agent that is produced endogenously and enzymatically by phagocytic cells Organic radicals are generated when superoxide or the hydroxyl radical indiscriminately extract electrons from other molecules Organic peroxy radicals are intermediates of chain reactions, such as lipid peroxidation Other organic radicals, such as the ethoxy radical, are intermediates of enzymatic reactions that escape into solution (see Table 24.2) An additional group of oxygen-containing radicals, termed RNOS, contain nitrogen as well as oxygen These are derived principally from the free radical nitric oxide (NO), which is produced endogenously by the enzyme nitric oxide synthase Nitric oxide combines with O2 or superoxide to produce additional RNOS C Major Sources of Primary Reactive Oxygen Species in the Cell ROS are constantly being formed in the cell; approximately to 5% of the oxygen we consume is converted to oxygen free radicals Some are produced as accidental by-products of normal enzymatic reactions that escape from the active site of metal-containing enzymes during oxidation reactions Others, such as hydrogen peroxide, are physiologic products of oxidases in peroxisomes Deliberate production of toxic free radicals occurs in the inflammatory response Drugs, natural radiation, air pollutants, and other chemicals also can increase formation of free radicals in cells CoQ GENERATES SUPEROXIDE One of the major sites of superoxide generation is Coenzyme Q (CoQ) in the mitochondrial electron transport chain (Fig 24.5) The one-electron reduced form of CoQ (CoQH•) is free within the membrane and can accidentally transfer an electron to dissolved O2, thereby forming superoxide In contrast, when O2 binds to cytochrome oxidase and accepts electrons, none of the O2 radical intermediates are released from the enzyme, and no ROS are generated CHAPTER 24 / OXYGEN TOXICITY AND FREE RADICAL INJURY With insufficient oxygen, Cora Nari’s ischemic heart muscle mitochondria were unable to maintain cellular ATP levels, resulting in high intracellular Naϩ and Ca2ϩ levels The reduced state of the electron carriers in the absence of oxygen, and loss of mitochondrial ion gradients or membrane integrity, leads to increased superoxide production once oxygen becomes available during reperfusion The damage can be self-perpetuating, especially if iron bound to components of the electron transport chain becomes available for the Fenton reaction, or the mitochondrial permeability transition is activated 443 NAD+ NADH NADH dehydrogenase FMN/ Fe–S O2 CoQH • CoQ – O2 Most of the oxidases, peroxidases, and oxygenases in the cell bind O2 and transfer single electrons to it via a metal Free radical intermediates of these reactions may be accidentally released before the reduction is complete Cytochrome P450 enzymes are a major source of free radicals “leaked” from reactions Because these enzymes catalyze reactions in which single electrons are transferred to O2 and an organic substrate, the possibility of accidentally generating and releasing free radical intermediates is high (see Chapters 19 and 25) Induction of P450 enzymes by alcohol, drugs, or chemical toxicants leads to increased cellular injury When substrates for cytochrome P450 enzymes are not present, its potential for destructive damage is diminished by repression of gene transcription Hydrogen peroxide and lipid peroxides are generated enzymatically as major reaction products by a number of oxidases present in peroxisomes, mitochondria, and the endoplasmic reticulum For example, monoamine oxidase, which oxidatively degrades the neurotransmitter dopamine, generates H2O2 at the mitochondrial membrane of certain neurons Peroxisomal fatty acid oxidase generates H2O2 rather than FAD(2H) during the oxidation of very-long-chain fatty acids (see Chapter 23) Xanthine oxidase, an enzyme of purine degradation that can reduce O2 to O2Ϫor H2O2 in the cytosol, is thought to be a major contributor to ischemia–reperfusion injury, especially in intestinal mucosal and endothelial cells Lipid peroxides are also formed enzymatically as intermediates in the pathways for synthesis of many eicosanoids, including leukotrienes and prostaglandins Fe – S OXIDASES, OXYGENASES, AND PEROXIDASES IONIZING RADIATION Cosmic rays that continuously bombard the earth, radioactive chemicals, and xrays are forms of ionizing radiation Ionizing radiation has a high enough energy level that it can split water into the hydroxyl and hydrogen radicals, thus leading to radiation damage to the skin, mutations, cancer, and cell death (Fig 24.6) It also may generate organic radicals through direct collision with organic cellular components Production of ROS by xanthine oxidase in endothelial cells may be enhanced during ischemia–reperfusion in Cora Nari’s heart In undamaged tissues, xanthine oxidase exists as a dehydrogenase that uses NADϩ rather than O2 as an electron acceptor in the pathway for degradation of purines (hypoxanthine xanthine uric acid (see Chapter 41) When O2 levels decrease, phosphorylation of ADP to ATP decreases, and degradation of ADP and adenine through xanthine oxidase increases In the process, xanthine dehydrogenase is converted to an oxidase As long as O2 levels are below the high Km of the enzyme for O2, little damage is done However, during reperfusion when O2 levels return to normal, xanthine oxidase generates H2O2 and O2Ϫ at the site of injury Cytochrome b – c1, Fe-H Fe-H c O2 H2O Fe-H– Cu Cytochrome aa3 Fig 24.5 Generation of superoxide by CoQ in the electron transport chain In the process of transporting electrons to O2, some of the electrons escape when CoQH• accidentally interacts with O2 to form superoxide Fe-H represents the Fe-heme center of the cytochromes Carbon tetrachloride (CCl4), which is used as a solvent in the dry-cleaning industry, is converted by cytochrome P450 to a highly reactive free radical that has caused hepatocellular necrosis in workers When the enzyme-bound CCl4 accepts an electron, it dissociates into CCl3· and Cl· The CCl3· radical, which cannot continue through the P450 reaction sequence, “leaks” from the enzyme active site and initiates chain reactions in the surrounding polyunsaturated lipids of the endoplasmic reticulum These reactions spread into the plasma membrane and to proteins, eventually resulting in cell swelling, accumulation of lipids, and cell death Les Dopaman, who is in the early stages of Parkinson’s disease, is treated with a monoamine oxidase B inhibitor Monoamine oxidase is a coppercontaining enzyme that inactivates dopamine in neurons, producing H2O2 The drug was originally administered to inhibit dopamine degradation However, current theory suggests that the effectiveness of the drug is also related to decrease of free radical formation within the cells of the basal ganglia The dopaminergic neurons involved are particularly susceptible to the cytotoxic effects of ROS and RNOS that may arise from H2O2 444 SECTION FOUR / FUEL OXIDATION AND THE GENERATION OF ATP H2O Ionizing radiation hv •OH Hydroxyl radical + H• Hydrogen atom Fig 24.6 Generation of free radicals from radiation The appearance of lipofuscin granules in many tissues increases during aging The pigment lipofuscin (from the Greek “lipos” for lipids and the Latin “fuscus” for dark) consists of a heterogeneous mixture of cross-linked polymerized lipids and protein formed by reactions between amino acid residues and lipid peroxidation products, such as malondialdehyde These cross-linked products are probably derived from peroxidatively damaged cell organelles that were autophagocytized by lysosomes but could not be digested When these dark pigments appear on the skin of the hands in aged individuals, they are referred to as “liver spots,” a traditional hallmark of aging In Les Dopaman and other patients with Parkinson’s disease, lipofuscin appears as Lewy bodies in degenerating neurons Evidence of protein damage shows up in many diseases, particularly those associated with aging In patients with cataracts, proteins in the lens of the eye exhibit free radical damage and contain methionine sulfoxide residues and tryptophan degradation products II OXYGEN RADICAL REACTIONS WITH CELLULAR COMPONENTS Oxygen radicals produce cellular dysfunction by reacting with lipids, proteins, carbohydrates, and DNA to extract electrons (summarized in Fig 24.7) Evidence of free radical damage has been described in over 100 disease states In some of these diseases, free radical damage is the primary cause of the disease; in others, it enhances complications of the disease A Membrane Attack: Formation of Lipid and Lipid Peroxy Radicals Chain reactions that form lipid free radicals and lipid peroxides in membranes make a major contribution to ROS-induced injury (Fig 24.8) An initiator (such as a hydroxyl radical produced locally in the Fenton reaction) begins the chain reaction It extracts a hydrogen atom, preferably from the double bond of a polyunsaturated fatty acid in a membrane lipid The chain reaction is propagated when O2 adds to form lipid peroxyl radicals and lipid peroxides Eventually lipid degradation occurs, forming such products as malondialdehyde (from fatty acids with three or more double bonds), and ethane and pentane (from the -terminal carbons of and fatty acids, respectively) Malondialdehyde appears in the blood and urine and is used as an indicator of free radical damage Peroxidation of lipid molecules invariably changes or damages lipid molecular structure In addition to the self-destructive nature of membrane lipid peroxidation, the aldehydes that are formed can cross-link proteins When the damaged lipids are the constituents of biologic membranes, the cohesive lipid bilayer arrangement and stable structural organization is disrupted (see Fig 24.7) Disruption of mitochondrial membrane integrity may result in further free radical production Respiratory enzymes Protein damage Mitochondrial damage Membrane damage SER RER DNA damage Nucleus (DNA) DNA O2– OH• H2O Na+ Ca Cell swelling 2+ Increased permeability Massive influx of Ca2+ Lipid peroxidation Fig 24.7 Free radical–mediated cellular injury Superoxide and the hydroxyl radical initiate lipid peroxidation in the cellular, mitochondrial, nuclear, and endoplasmic reticulum membranes The increase in cellular permeability results in an influx of Ca2 ϩ , which causes further mitochondrial damage The cysteine sulfhydryl groups and other amino acid residues on proteins are oxidized and degraded Nuclear and mitochondrial DNA can be oxidized, resulting in strand breaks and other types of damage RNOS (NO, NO2, and peroxynitrite) have similar effects CHAPTER 24 / OXYGEN TOXICITY AND FREE RADICAL INJURY B Proteins and Peptides In proteins, the amino acids proline, histidine, arginine, cysteine, and methionine are particularity susceptible to hydroxyl radical attack and oxidative damage As a consequence of oxidative damage, the protein may fragment or residues cross-link with other residues Free radical attack on protein cysteine residues can result in cross-linking and formation of aggregates that prevents their degradation However, oxidative damage increases the susceptibility of other proteins to proteolytic digestion Free radical attack and oxidation of the cytsteine sulfhydryl residues of the tripeptide glutathione (␥-glutamyl-cysteinyl-glycine; see section V.A.3.) increases oxidative damage throughout the cell Glutathione is a major component of cellular defense against free radical injury, and its oxidation reduces its protective effects 445 A Initiation LH + •OH L • + OH y • x L• B Propagation L• + O2 LOO • + LOO • LOOH + L • LH • O O y x C DNA Oxygen-derived free radicals are also a major source of DNA damage Approximately 20 types of oxidatively altered DNA molecules have been identified The nonspecific binding of Fe2ϩ to DNA facilitates localized production of the hydroxyl radical, which can cause base alterations in the DNA (Fig 24.9) It also can attack the deoxyribose backbone and cause strand breaks This DNA damage can be repaired to some extent by the cell (see Chapter 12), or minimized by apoptosis of the cell LOO • H O O y x Lipid peroxide LOOH III NITRIC OXIDE AND REACTIVE NITROGEN-OXYGEN SPECIES (RNOS) Nitric oxide (NO) is an oxygen-containing free radical which, like O2, is both essential to life and toxic NO has a single electron, and therefore binds to other compounds containing single electrons, such as Fe3ϩ As a gas, it diffuses through the cytosol and lipid membranes and into cells At low concentrations, it functions physiologically as a neurotransmitter and a hormone that causes vasodilation However, at high concentrations, it combines with O2 or with superoxide to form additional reactive and toxic species containing both nitrogen and oxygen (RNOS) RNOS are involved in neurodegenerative diseases, such as Parkinson’s disease, and in chronic inflammatory diseases, such as rheumatoid arthritis C Degradation y O + O Malondialdehyde Degraded lipid peroxide D Termination LOO • + Nitroglycerin, in tablet form, is often given to patients with coronary artery disease who experience ischemia-induced chest pain (angina) The nitroglycerin decomposes in the blood, forming NO, a potent vasodilator, which increases blood flow to the heart and relieves the angina LOOH + LH L• A Nitric Oxide Synthase At low concentrations, nitric oxide serves as a neurotransmitter or a hormone It is synthesized from arginine by nitric oxide synthases (Fig 24.10) As a gas, it is able to diffuse through water and lipid membranes, and into target cells In the target cell, it exerts its physiologic effects by high-affinity binding to Fe-heme in the enzyme guanylyl cyclase, thereby activating a signal transduction cascade However, NO is rapidly inactivated by nonspecific binding to many molecules, and therefore cells that produce NO need to be close to the target cells The body has three different tissue-specific isoforms of NO synthase, each encoded by a different gene: neuronal nitric oxide synthase (nNOS, isoform I), inducible nitric oxide synthase (iNOS, isoform II), and endothelial nitric oxide synthase (eNOS, isoform III) nNOS and eNOS are tightly regulated by Ca2ϩ concentration to produce the small amounts of NO required for its role as a neurotransmitter and hormone In contrast, iNOS is present in many cells of the immune system and cell types with a similar lineage, such as macrophages and x O O H or L• + Vit E Vit E• + L• LH + Vit E• LH + Vit EOX Fig 24.8 Lipid peroxidation: a free radical chain reaction A Lipid peroxidation is initiated by a hydroxyl or other radical that extracts a hydrogen atom from a polyunsaturated lipid (LH), thereby forming a lipid radical (L•) B The free radical chain reaction is propagated by reaction with O2, forming the lipid peroxy radical (LOO•) and lipid peroxide (LOOH) C Rearrangements of the single electron result in degradation of the lipid Malondialdehyde, one of the compounds formed, is soluble and appears in blood D The chain reaction can be terminated by vitamin E and other lipid-soluble antioxidants that donate single electrons Two subsequent reduction steps form a stable, oxidized antioxidant 446 SECTION FOUR / FUEL OXIDATION AND THE GENERATION OF ATP brain astroglia This isoenzyme of nitric oxide synthase is regulated principally by induction of gene transcription, and not by changes in Ca2ϩ concentration It produces high and toxic levels of NO to assist in killing invading microorganisms It is these very high levels of NO that are associated with generation of RNOS and NO toxicity O C N N N H HN H2N Guanine B NO Toxicity The toxic actions of NO can be divided into two categories: direct toxic effects resulting from binding to Fe-containing proteins, and indirect effects mediated by compounds formed when NO combines with O2 or with superoxide to form RNOS •OH O C HN N OH H2N N N H 8-hydroxyguanine Fig 24.9 Conversion of guanine to 8-hydroxyguanine by the hydroxy radical The amount of 8-hydroxyguanosine present in cells can be used to estimate the amount of oxidative damage they have sustained The addition of the hydroxyl group to guanine allows it to mispair with T residues, leading to the creation of a daughter molecule with an A-T base pair in this position DIRECT TOXIC EFFECTS OF NO NO, as a radical, exerts direct toxic effects by combining with Fe-containing compounds that also have single electrons Major destructive sites of attack include FeS centers (e.g., electron transport chain complexes I-III, aconitase) and Fe-heme proteins (e.g., hemoglobin and electron transport chain cytochromes) However, there is usually little damage because NO is present in low concentrations and Feheme compounds are present in excess capacity NO can cause serious damage, however, through direct inhibition of respiration in cells that are already compromised through oxidative phosphorylation diseases or ischemia RNOS TOXICITY When present in very high concentrations (e.g., during inflammation), NO combines nonenzymatically with superoxide to form peroxynitrite (ONOOϪ ), or with O2 to form N2O3 (Fig 24.11) Peroxynitrite, although not a free radical, is a strong Arginine Nitric oxide synthase NO• O2 NO• NO2 NO• Nitric oxide (free radical) Citrulline N2O3 Nitrogen trioxide (nitrosating agent) O2– NO• ONOO– NO2– Peroxynitrite (strong oxidizing agent) Nitrite physiologic pH H+ Arginine HONO2 NADPH FORMS OF RNOS Diet, Intestinal bacteria Peroxynitrous acid O2 NO synthase (Fe-Heme, FAD, FMN) NO Nitric oxide NADP+ Citrulline Fig 24.10 Nitric oxide synthase synthesizes the free radical NO Like cytochrome P450 enzymes, NO synthase uses Fe-heme, FAD, and FMN to transfer single electrons from NADPH to O2 NO3– Nitrate ion (safe) OH– + NO2+ •OH Hydroxyl radical + Nitronium ion (nitrating agent) NO2• Nitrogen dioxide (free radical) Smog Organic smoke Cigarettes Fig 24.11 Formation of RNOS from nitric oxide RNOS are shown in blue The type of damage caused by each RNOS is shown in parentheses Of all the nitrogen–oxygen-containing compounds shown, only nitrate is relatively nontoxic CHAPTER 24 / OXYGEN TOXICITY AND FREE RADICAL INJURY oxidizing agent that is stable and directly toxic It can diffuse through the cell and lipid membranes to interact with a wide range of targets, including protein methionine and -SH groups (e.g., Fe-S centers in the electron transport chain) It also breaks down to form additional RNOS, including the free radical nitrogen dioxide (NO2), an effective initiator of lipid peroxidation Peroxynitrite products also nitrate aromatic rings, forming compounds such as nitrotyrosine or nitroguanosine N2O3, which can be derived either from NO2 or nitrite, is the agent of nitrosative stress, and nitrosylates sulfhydryl and similarily reactive groups in the cell Nitrosylation will usually interefere with the proper functioning of the protein or lipid that has been modified Thus, RNOS can as much oxidative and free radical damage as non–nitrogen-containing ROS, as well as nitrating and nitrosylating compounds The result is widespread and includes inhibition of a large number of enzymes; mitochondrial lipid peroxidation; inhibition of the electron transport chain and energy depletion; single-stranded or double-stranded breaks in DNA; and modification of bases in DNA 447 NO2 is one of the toxic agents present in smog, automobile exhaust, gas ranges, pilot lights, cigarette smoke, and smoke from forest fires or burning buildings IV FORMATION OF FREE RADICALS DURING PHAGOCYTOSIS AND INFLAMMATION In response to infectious agents and other stimuli, phagocytic cells of the immune system (neutrophils, eosinophils, and monocytes/macrophages) exhibit a rapid consumption of O2 called the respiratory burst The respiratory burst is a major source of superoxide, hydrogen peroxide, the hydroxyl radical, hypochlorous acid (HOCl), and RNOS The generation of free radicals is part of the human antimicrobial defense system and is intended to destroy invading microorganisms, tumor cells, and other cells targeted for removal A NADPH Oxidase The respiratory burst results from the activity of NADPH oxidase, which catalyzes the transfer of an electron from NADPH to O2 to form superoxide (Fig 24.12) NADPH oxidase is assembled from cytosol and membranous proteins recruited into the phagolysosome membrane as it surrounds an invading microorganism Superoxide is released into the intramembranous space of the phagolysosome, where it is generally converted to hydrogen peroxide and other ROS that are effective against bacteria and fungal pathogens Hydrogen peroxide is formed by superoxide dismutase, which may come from the phagocytic cell or the invading microorganism B Myeloperoxidase and HOCl The formation of hypochlorous acid from H2O2 is catalyzed by myeloperoxidase, a heme-containing enzyme that is present only in phagocytic cells of the immune system (predominantly neutrophils) Myeloperoxidase Dissociation H2O2 ϩ ClϪ ϩ Hϩ S HOCl ϩ H2O S ϪOCl ϩ Hϩ ϩ H2O Myeloperoxidase contains two Fe heme-like centers, which give it the green color seen in pus Hypochlorous acid is a powerful toxin that destroys bacteria within seconds through halogenation and oxidation reactions It oxidizes many Fe and S-containing groups (e.g., sulfhydryl groups, iron-sulfur centers, ferredoxin, heme-proteins, methionine), oxidatively decarboxylates and deaminates proteins, and cleaves peptide bonds Aerobic bacteria under attack rapidly lose membrane In patients with chronic granulomatous disease, phagocytes have genetic defects in NADPH oxidase NADPH oxidase has four different subunits (two in the cell membrane and two recruited from the cytosol), and the genetic defect may be in any of the genes that encode these subunits The membrane catalytic subunit  of NADPH oxidase is a 91-kDa flavocytochrome glycoprotein It transfers electrons from bound NADPH to FAD, which transfers them to the Fe–heme components The membranous ␣-subunit (p22) is required for stabilization Two additional cytosolic proteins (p47phox and p67phox) are also required for assembly of the complex Rac, a monomeric GTPase in the Ras subfamily of the Rho superfamily (see Chapter 9), is also required for assembly The 91-kDa subunit is affected most often in X-linked chronic granulatomous disease, whereas the ␣-subunit is affected in a rare autosomal recessive form The cytosolic subunits are affected most often in patients with the autosomal recessive form of granulomatous disease In addition to their enhanced susceptibility to bacterial and fungal infections, these patients suffer from an apparent dysregulation of normal inflammatory responses 448 SECTION FOUR / FUEL OXIDATION AND THE GENERATION OF ATP NADPH O2 NADPH oxidase – NADP+ O2 NO Bacterium H2O2 HOCL iNOS Fe2+ Cl– Fe3+ myeloperoxidase OH • ONOO– Bacterium Invagination of neutrophil's cytoplasmic membrane Fig 24.12 Production of reactive oxygen species during the phagocytic respiratory burst by activated neutrophils (1) Activation of NADPH oxidase on the outer side of the plasma membrane initiates the respiratory burst with the generation of superoxide During phagocytosis, the plasma membrane invaginates, so superoxide is released into the vacuole space (2) Superoxide (either spontaneously or enzymatically via superoxide dismutase [SOD]) generates H2O2 (3) Granules containing myeloperoxidase are secreted into the phagosome, where myeloperoxidase generates HOCl and other halides (4) H2O2 can also generate the hydroxyl radical from the Fenton reaction (5) Inducible nitric oxide synthase may be activated and generate NO (6) Nitric oxide combines with superoxide to form peroxynitrite, which may generate additional RNOS The result is an attack on the membranes and other components of phagocytosed cells, and eventual lysis The whole process is referred to as the respiratory burst because it lasts only 30 to 60 minutes and consumes O2 transport, possibly because of damage to ATP synthase or electron transport chain components (which reside in the plasma membrane of bacteria) C RNOS and Inflammation During Cora Nari’s ischemia (decreased blood flow), the ability of her heart to generate ATP from oxidative phosphorylation was compromised The damage appeared to accelerate when oxygen was first reintroduced (reperfused) into the tissue During ischemia, CoQ and the other single-electron components of the electron transport chain become saturated with electrons When oxygen is reintroduced (reperfusion), electron donation to O2 to form superoxide is increased The increase of superoxide results in enhanced formation of hydrogen peroxide and the hydroxyl radical Macrophages in the area to clean up cell debris from ischemic injury produce nitric oxide, which may further damage mitochondria by generating RNOS that attack Fe-S centers and cytochromes in the electron transport chain membrane lipids Thus, the RNOS may increase the infarct size When human neutrophils of the immune system are activated to produce NO, NADPH oxidase is also activated NO reacts rapidly with superoxide to generate peroxynitrite, which forms additional RNOS NO also may be released into the surrounding medium, to combine with superoxide in target cells In a number of disease states, free radical release by neutrophils or macrophages during an inflammation contributes to injury in the surrounding tissues During stroke or myocardial infarction, phagocytic cells that move into the ischemic area to remove dead cells may increase the area and extent of damage The selfperpetuating mechanism of radical release by neutrophils during inflammation and immune complex formation may explain some of the features of chronic inflammation in patients with rheumatoid arthritis As a result of free radical release, the immunoglobulin G (IgG) proteins present in the synovial fluid are partially oxidized, which improves their binding with the rheumatoid factor antibody This binding, in turn, stimulates the neutrophils to release more free radicals V CELLULAR DEFENSES AGAINST OXYGEN TOXICITY Our defenses against oxygen toxicity fall into the categories of antioxidant defense enzymes, dietary and endogenous antioxidants (free radical scavengers), cellular compartmentation, metal sequestration, and repair of damaged cellular components The antioxidant defense enzymes react with ROS and cellular products of free radical chain reactions to convert them to nontoxic products Dietary antioxidants, such as vitamin E and flavonoids, and endogenous antioxidants, such as urate, can 906 SECTION EIGHT / TISSUE METABOLISM Cell movement within the extracellular matrix requires remodeling of the various components of the matrix This is accomplished by a variety of matrix metalloproteinases (MMPs) and regulators of the MMPs, tissue inhibitors of matrix metalloproteinases (TIMPs) Dysregulation of this delicate balance of the regulators of cell movement allows cancer cells to travel to other parts of the body (metastasize) as well as to spread locally to contiguous tissues THE WAITING ROOM Sis Lupus (first introduced in Chapter 14) noted a moderate reduction in pain and swelling in the joints of her fingers when she was taking a 6-week course of high-dose prednisone, an anti-inflammatory steroid As the dose of this drug was tapered to minimize its long-term side effects, however, the pain in the joints of her fingers returned, and, for the first time, her left knee became painful, swollen, and warm to the touch Her rheumatologist described to her the underlying inflammatory tissue changes that her systemic lupus erythematosus (SLE) was causing in the joint tissues Ann Sulin complained of a declining appetite for food as well as severe weakness and fatigue The reduction in her kidneys’ ability to maintain normal daily total urinary net acid excretion contributed to her worsening metabolic acidosis This plus her declining ability to excrete nitrogenous waste products, such as creatinine and urea, into her urine (“azotemia”) are responsible for many of her symptoms Her serum creatinine level was rising steadily As it approached a level of mg/dL, she developed a litany of complaints caused by the multisystem dysfunction associated with her worsening metabolic acidosis, retention of nitrogenous waste products, and so forth (“uremia”) Her physicians discussed with Ann the need to consider peritoneal dialysis or hemodialysis I COMPOSITION OF THE EXTRACELLULAR MATRIX A Fibrous Proteins Fig 49.2 The triple helix of collagen COLLAGEN Collagen, a family of fibrous proteins, is produced by a variety of cell types but principally by fibroblasts (cells found in interstitial connective tissue), muscle cells, and epithelial cells Type I collagen [collagen(I)], the most abundant protein in mammals, is a fibrous protein that is the major component of connective tissue It is found in the extracellular matrix (ECM) of loose connective tissue, bone, tendons, skin, blood vessels, and the cornea of the eye Collagen(I) contains approximately 33% glycine and 21% proline and hydroxyproline Hydroxyproline is an amino acid produced by posttranslational modification of peptidyl proline residues (see Chapter 7, section V.C., for an earlier introduction to collagen) Procollagen(I), the precursor of collagen(I), is a triple helix composed of three polypeptide (pro-␣) chains that are twisted around each other, forming a rope-like structure Polymerization of collagen(I) molecules forms collagen fibrils, which provide great tensile strength to connective tissues (Fig 49.2) The individual polypeptide chains each contain approximately 1,000 amino acid residues The three polypeptide chains of the triple helix are linked by interchain hydrogen bonds CHAPTER 49 / THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE Each turn of the triple helix contains three amino acid residues, such that every third amino acid is in close contact with the other two strands in the center of the structure Only glycine, which lacks a side chain, can fit in this position, and indeed, every third amino acid residue of collagen is glycine Thus, collagen is a polymer of (Gly-X-Y) repeats, where Y is frequently proline or hydroxyproline, and X is any other amino acid found in collagen Procollagen(I) is an example of a protein that undergoes extensive posttranslational modifications Hydroxylation reactions produce hydroxyproline residues from proline residues and hydroxylysine from lysine residues These reactions occur after the protein has been synthesized (Fig 49.3) and require vitamin C (ascorbic acid) as a cofactor of the enzymes, for example, prolyl hydroxylases and lysyl hydroxylase Hydroxyproline residues are involved in hydrogen bond formation that helps to stabilize the triple helix, whereas hydroxylysine residues are the sites of attachment of disaccharide moieties (galactose-glucose) The side chains of lysine residues also may be oxidized to form the aldehyde, allysine These aldehyde residues produce covalent cross-links between collagen molecules (Fig 49.4) An allysine residue on one collagen molecule reacts with the amino group of a lysine residue on another molecule, forming a covalent Schiff base that is converted to more stable covalent cross-links Aldol condensation also may occur between two allysine residues, which forms the structure lysinonorleucine 907 The role of carbohydrates in collagen structure is still controversial The hydroxyproline residues in collagen are required for stabilization of the triple helix by hydrogen bond formation In the absence of vitamin C (scurvy), the melting temperature of collagen can drop from 42oC to 24oC, because of the loss of interstrand hydrogen bond formation from the lack of hydroxyproline residues i Types of Collagen At least 19 different types of collagen have been characterized (Table 49.1) Although each type of collagen is found only in particular locations in the body, more than one type may be present in the ECM at a given location The various types of collagen can be classified as fibril-forming (types I, II, III, V, and XI), network-forming (types IV, VIII and X), those that associate with fibril surfaces (types IX, XII, and XIV), those that are transmembrane proteins (types XIII and XVII), endostatin-forming (types XV and XVIII), and those that form periodic beaded filaments (type VI) O N CH H 2C O C N + α -Ketoglutarate CH2 prolyl hydroxylase H H2C Ascorbate C O2 H CH CH2 CO2 H OH - Hydroxyproline residue O H CH CH2 CH2 CH2 O C + α -Ketoglutarate lysyl hydroxylase Ascorbate O2 CO2 N CH H CH2 C + Succinate CH2 CH CH2 CH2 + NH + NH Lysine residue + Succinate C Proline residue N C OH - Hydroxylysine residue Fig 49.3 Hydroxylation of proline and lysine residues in collagen Proline and lysine residues within the collagen chains are hydroxylated by reactions that require vitamin C Protein 908 SECTION EIGHT / TISSUE METABOLISM δ ε Table 49.1 Types of Collagen + CH2 CH2 NH3 Collagen Type Lysine residue A Gene Structural Details Localization I II III Col1A1-Col1A2 Col2A1 Col3A1 Fibrils Fibrils Fibrils IV Col4A1–Col4A6 Nonfibrillar, mesh collagen V Col5A1-Col5A3 Small fibers, N-terminal globular domains VI Col6A1-Col6A3 VII Col7A1 Microfibrils, with both N and C-terminal globular domains An anchoring collagen VIII Col8A1-Col8A2 Nonfibrillar, mesh collagen IX Col9A1-Col9A3 X Col10A1 XI XII Col11A1-Col11A3 Col12A1 Fibril-associated collagens with interrupted triple helices (FACIT); N-terminal globular domain Nonfibrillar, mesh collagen, with C-terminal globular domain Small fibers FACIT Skin, tendon, bone, cornea Cartilage, vitreous humour Skin, muscle, associates with type I collagen All basal laminae (basement membranes) Associates with type I collagen in most interstitial tissues Associates with type I collagen in most interstitial tissues Epithelial cells; dermal– epidermal junction Cornea, some endothelial cells Associates with type II collagen in cartilage and vitreous humour XIII XIV XV XVI XVII XVIII XIX Col13A1 Col14A1 Col15A1 Col16A1 Col17A1 Col18A1 Col19A1 Transmembrane collagen FACIT Endostatin-forming collagen Other Transmembrane collagen Endostatin-forming Other O2 lysyl oxidase NH3 + OH – ε O C H δ CH2 + ε H2N δ CH2 CH2 Second lysine residue Allysine residue B H2O δ CH2 ε CH ε N CH2 δ CH2 Schiff base δ CH2 ε O C H HO ε + C H Allysine (aldo form) C δ CH Allysine (enol form) Aldol condensation δ CH2 δ Cartilage, vitreous humor Interacts with types I and II collagen in soft tissues Cell surfaces, epithelial cells Soft tissue Endothelial cells Ubiquitous Epidermal cell surface Endothelial cells Ubiquitous See the text for descriptions of the differences in types of collagen HO ε H ε O C CH δ CH H2O CH2 Growth plate, hypertrophic and mineralizing cartilage ε H ε O C CH C δ Lysinonorleucine Fig 49.4 Formation of cross-links in collagen A Lysine residues are oxidized to allysine (an aldehyde) Allysine may react with an unmodified lysine residue to form a Schiff base (B), or two allysine residues may undergo an aldol condensation (C) All collagens contain three polypeptide chains with at least one stretch of triple helix The non–triple helical domains can be short (such as in the fibril-forming collagens) or can be rather large, such that the triple helix is actually a minor component of the overall structure (examples are collagen types XII and XIV) The FACIT (fibril-associated collagens with interrupted triple helices, collagen types IX, XII, and XIV) collagen types associate with fibrillar collagens, without themselves forming fibers The endostatin-forming collagens are cleaved at their C-terminus to form endostatin, an inhibitor of angiogenesis The network-forming collagens (type IV) form a mesh-like structure, because of large (approximately 230 amino acids) noncollagenous domains at the carboxy-terminal (Fig 49.5) And finally, a number of collagen types are actually transmembrane proteins (XIII and XVII) found on epithelial or epidermal cell surfaces, which play a role in a number of cellular processes, including adhesion of components of the ECM to cells embedded within it Types I, II, and III collagens form fibrils that assemble into large insoluble fibers The fibrils (see below) are strengthened through covalent cross-links between lysine residues on adjacent fibrils The arrangement of the fibrils gives individual tissues their distinct characteristics Tendons, which attach muscles to bones, contain collagen Endostatins block angiogenesis (new blood vessel formation) by inhibiting endothelial cell migration Because endothelial cell migration and proliferation are required to form new blood vessels, inhibiting this action blocks angiogenesis Tumor growth is dependent on a blood supply; inhibiting angiogenesis can reduce tumor cell proliferation CHAPTER 49 / THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE 909 A Protomer Carboxy terminal Amino terminal B Dimer Carboxy terminal hexamer (NC1 domain) C Type IV collagen tetramer Aggregation at amino termini (7S domain) D Suprastructure NC1 hexamer 7S domain Fig 49.5 Type IV collagen contains a globular carboxy-terminal domain (A), which forms tropocollagen dimers (hexamers of collagen, B) Four dimers associate at the amino-terminal domains to form a 7S domain (C), and the tetramers form a lattice (D), which provides structural support to the basal lamina fibrils aligned parallel to the long axis of the tendon, thus giving the tendon tremendous tensile strength The types of collagen that not form fibrils perform a series of distinct roles Fibril-associated collagens bind to the surface of collagen fibrils and link them to other matrix- forming components The transmembrane collagens form anchoring fibrils that link components of the extracellular matrix to underlying connective tissue The network- forming collagens (type IV) form a flexible collagen that is part of the basement membrane and basal lamina that surround many cells ii Synthesis and Secretion of Collagen Collagen is synthesized within the endoplasmic reticulum as a precursor known as preprocollagen The presequence acts as the signal sequence for the protein and is cleaved, forming procollagen within the endoplasmic reticulum From there it is transported to the Golgi apparatus (Table 49.2) Three procollagen molecules associate through formation of intrastrand disulfide bonds at the carboxy-terminus; once One type of osteogenesis imperfecta (OI) is caused by a mutation in a gene that codes for collagen The phenotype of affected individuals varies greatly, depending on the location and type of mutation See the Biochemical Comments for more information concerning this type of OI 910 SECTION EIGHT / TISSUE METABOLISM Table 49.2 Steps Involved in Collagen Biosynthesis Location Rough endoplasmic reticulum Lumen of the ER Lumen of ER and Golgi apparatus Secretory vesicle Extracellular Process Synthesis of preprocollagen; insertion of the procollagen molecule into the lumen of the ER Hyroxylation of proline and lysine residues; glycosylation of selected hydroxylysine residues Self-assembly of the tropocollagen molecule, initiated by disulfide bond formation in the carboxy-terminal extensions; triple helix formation Procollagen prepared for secretion from cell Cleavage of the propeptides, removing the amino- and carboxy-terminal extensions, and self-assembly of the collagen molecules into fibrils, and then fibers these disulfides are formed, the three molecules can align properly to initiate formation of the triple helix The triple helix forms from the carboxy-end toward the amino-end, forming tropocollagen The tropocollagen contains a triple helical segment between two globular ends, the amino- and carboxy-terminal extensions The tropocollagen is secreted from the cell, the extensions are removed using extracellular proteases, and the mature collagen takes its place within the ECM The individual fibrils of collagen line up in a highly ordered fashion to form the collagen fiber ELASTIN Elastin is the major protein found in elastic fibers, which are located in the ECM of connective tissue of smooth muscle cells, endothelial and microvascular cells, chondrocytes, and fibroblasts Elastic fibers allow tissues to expand and contract; this is of particular importance to blood vessels, which must deform and reform repeatedly in response to the changes in intravascular pressure that occur with the contraction of the left ventricle of the heart It is also important for the lungs, which stretch each time a breath is inhaled and return to their original shape with each exhalation In addition to elastin, the elastic fibers contain microfibrils, which are composed of a number of acidic glycoproteins, the major ones being fibrillin-1 and fibrillin-2 i Tropoelastin Elastin has a highly cross-linked, insoluble, amorphous structure Its precursor, tropoelastin, is a molecule of high solubility, which is synthesized on the rough endoplasmic reticulum (RER) for eventual secretion Tropoelastin contains two types of alternating domains The first domain consists of a hydrophilic sequence rich in lysine and alanine residues The second domain consists of a hydrophobic sequence rich in valine, proline, and glycine, which frequently occur in repeats of VPGVG or VGGVG The protein contains approximately 16 regions of each domain, alternating throughout the protein (Fig 49.6) Hydrophilic C-terminal Signal peptide Hydrophilic cross-linking exons Hydrophilic exon 26a (for alternative splicing) 3'-untranslated region Hydrophobic exons Fig 49.6 The cDNA structure of elastin, indicating the repeating cross-linking and hydrophobic domains CHAPTER 49 / THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE On secretion from the cell, the tropoelastin is aligned with the microfibrils, and lysyl oxidase initiates the reactions that cross-link elastin molecules, using lysine residues within the hydrophilic alternating domains in the proteins This cross-linking reaction is the same as that which occurs in collagen In this reaction, 2, 3, or lysine residues are cross- linked to form a stable structure The net result of the cross-linking is the generation of a fibrous mesh that encircles the cells ii Elastic Properties of Elastin Elastic fibers have the ability to stretch and then to reform without requiring an obvious energy source to so The mechanism by which this stretching and relaxing actively occurs is still controversial but does relate to the basic principles of protein folding described in Chapter When the elastic fibers are stretched (such as when a breath is taken in and the lung fills up with air), the amorphous elastin structure is stretched This stretching exposes the repeating hydrophobic regions of the molecule to the aqueous environment This, in turn, leads to a decrease in the entropy of water, because the water molecules need to rearrange to form cages about each hydrophobic domain When this stretching force within the lung is removed (e.g., when the subject exhales), the elastin takes on its original structure because of the increase in entropy that occurs because the water no longer needs to form cages about hydrophobic domains Thus, the hydrophobic effect is the primary force that allows this stretched structure to reform Elastin is inherently stable, with a half-life of up to 70 years LAMININ After type IV collagen, laminin is the most abundant protein in basal laminae Laminin provides additional structural support for the tissues through its ability to bind to type IV collagen, to other molecules present in the ECM, and to cell surface–associated proteins (the integrins, see section D) 911 Supravalvular aortic stenosis (SVAS) results from an insufficiency of elastin in the vessel wall, leading to a narrowing of the large elastic arteries Current theory suggests that the levels of elastin in the vessel walls may regulate the number of smooth muscle cell rings that encircle the vessel If the levels of elastin are low, smooth muscle hypertrophy results, leading to a narrowing and stenosis of the artery α-chain HNH Gobular domains H HN β-chain H NH γ-chain Disulfide bonds HOOC Coiled-coil; rigid rods COOH i Laminin Structure Laminin is a heterotrimeric protein shaped, for the most part, like a cross (Fig 49.7) The trimer is composed of ␣, , and ␥ subunits There are five possible ␣ proteins (designated ␣1–␣5), three different versions of the  subunit (1–3), and three different ␥ forms (␥1 – ␥3) Thus, there is a potential for the formation of as many as 45 different combinations of these three subunits However, only 12 have been discovered (designated laminins 1–12) Laminin 1, composed of ␣11␥1, is typical of this class of proteins The major feature of the laminin structure is a coiled ␣-helix, which joins the three subunits together and forms a rigid rod All three chains have extensions at the amino-terminal end Only the ␣ chain has a significant carboxy-terminal extension past the rod-like structure It is the laminin extensions that allow laminin to bind to other components within the ECM and to provide stability for the structure Components of the ECM that are bound by laminin include collagen, sulfated lipids, and proteoglycans ii Laminin Biosynthesis Like other secreted proteins, laminin is synthesized with a leader sequence targeting the three chains to the endoplasmic reticulum Chain association occurs within the Golgi apparatus before secretion from the cell After laminin is secreted by the cell, the amino terminal extensions promote self-association, as well as the binding to other ECM components Disulfide linkages are formed to stabilize the trimer, but there is much less posttranslational processing of laminin than there is of collagen and elastin COOH Fig 49.7 The structure of laminin Defects in the structures of laminin or laminin (proteins that contribute to the cohesion of the dermis and epidermis) lead to the disorder referred to as junctional epidermolysis bullosa (JEB) In this disorder, there can be severe spontaneous blistering of the skin and mucous membranes A severe form of the disease, JEB gravis, is often fatal early in life Death occurs as a result of epithelial blistering of the respiratory, digestive, and genitourinary systems Congenital muscular dystrophy (CMD) results from a defect in laminin 2, which is a component of the bridge that links the muscle cell cytoskeleton to the extracellular matrix Lack of this bridge triggers muscle cell apoptosis, which results in weakened muscles 912 SECTION EIGHT / TISSUE METABOLISM The ECM is not simply a glue that holds cells together; it also serves to keep cells from moving to other locations and to prevent large molecules and other particles, such as microorganisms, from reaching contiguous and distant cells This confining property of the matrix is medically important For example, infections spread, in part, because the infectious agent alters the “containing” capacity of the ECM Cancer cells that metastasize (migrate to other tissues) can so only by altering the integrity of the matrix Diseases such as rheumatoid arthritis (an autoimmune destruction of articular and periarticular tissues) and osteoarthritis (degenerative joint disease often associated with aging) involve damage to the functional capacity of the matrix Alterations in the structural characteristics of the matrix of the renal glomerulus may allow proteins to be excreted into the urine, an indication of inexorable decline in renal function Genetic defects may cause components of the matrix to be structurally and functionally abnormal, resulting in connective tissue disorders such as the EhlersDanlos syndrome (caused by a number of mutations that affect specific collagen genes) and Marfan’s syndrome (a defect in the protein, fibrillin, in which over 330 different mutations, many of which give rise to different phenotypes, have been identified) Deficiencies of lysosomal enzymes involved in normal degradation of molecules of the matrix result in diseases such as the mucopolysaccharidoses The principal components of the matrix of cartilage are collagen and proteoglycans, both of which are produced and degraded by the chondrocytes that are embedded in this matrix An autoimmune attack on articular proteins alters the balance between cartilage degradation and formation The resulting loss of cartilage organization accompanied by an inflammatory response is responsible for the symptoms experienced by Sis Lupus The collagen component forms a network of fine fibrils that give shape to the cartilage The proteoglycans embedded in the cartilage are responsible for its compressibility and its deformability B Proteoglycans The fibrous structural proteins of the ECM are embedded in gels formed from proteoglycans Proteoglycans consist of polysaccharides called glycosaminoglycans (GAG) linked to a core protein The GAGs are composed of repeating units of disaccharides One sugar of the disaccharide is either N-acetylglucosamine or Nacetylgalactosamine, and the second is usually acidic (either glucuronic acid or iduronic acid) These sugars are modified by the addition of sulfate groups to the parent sugar A proteoglycan may contain more than 100 GAG chains and consist of up to 95% oligosaccharide by weight The negatively charged carboxylate and sulfate groups on the proteoglycan bind positively charged ions and form hydrogen bonds with trapped water molecules, thereby creating a hydrated gel The gel provides a flexible mechanical support to the ECM The gel also acts as a filter that allows the diffusion of ions (e.g., Ca2ϩ ), H2O, and other small molecules, but slows diffusion of proteins and movement of cells Hyaluronan is the only GAG that occurs as a single long polysaccharide chain and is the only GAG that is not sulfated STRUCTURE AND FUNCTION OF THE PROTEOGLYCANS Proteoglycans are found in interstitial connective tissues, for example, the synovial fluid of joints, the vitreous humor of the eye, arterial walls, bone, cartilage, and cornea They are major components of the ECM in these tissues The proteoglycans interact with a variety of proteins in the matrix, such as collagen and elastin, fibronectin (which is involved in cell adhesion and migration), and laminin Proteoglycans are proteins that contain many chains of GAGs (formerly called mucopolysaccharides) Glycosaminoglycans are long, unbranched polysaccharides composed of repeating disaccharide units (Fig 49.8) The repeating disaccharides usually contain an iduronic or uronic acid and a hexosamine and are frequently sulfated Consequently, they carry a negative charge, are hydrated, and act as lubricants After synthesis, proteoglycans are secreted from cells; thus, they function extracellularly Because the long, negatively charged glycosaminoglycan chains repel each other, the proteoglycans occupy a very large space and act as “molecular sieves,” determining which substances enter or leave cells (Table 49.3) Their properties also give resilience and a degree of flexibility to substances such as cartilage, permitting compression and reexpansion of the molecule to occur At least seven types of glycosaminoglycans exist, which differ in the monosaccharides present in their repeating disaccharide units—chondroitin sulfate, dermatan sulfate, heparin, heparin sulfate, hyaluronic acid, and keratan sulfates I and II Except for hyaluronic acid, the glycosaminoglycans are linked to proteins, usually attached covalently to serine or threonine residues (Fig 49.9) Keratan sulfate I is attached to asparagine SYNTHESIS OF THE PROTEOGLYCANS The protein component of the proteoglycans is synthesized on the ER It enters the lumen of this organelle, where the initial glycosylations occur UDP-sugars serve as The long polysaccharide side chains of the proteoglycans in cartilage contain many anionic groups This high concentration of negative charges attracts cations that create a high osmotic pressure within cartilage, drawing water into this specialized connective tissue and placing the collagen network under tension At equilibrium, the resulting tension balances the swelling pressure caused by the proteoglycans The complementary roles of this macromolecular organization give cartilage its resilience Cartilage can thus withstand the compressive load of weight bearing and then reexpand to its previous dimensions when that load is relieved 913 CHAPTER 49 / THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE Table 49.3 Some Specific Functions of the Glycosaminoglycans and Proteoglycans Glycosaminoglycan Hyaluronic acid Chondroitin sulfate proteoglycans Keratan sulfate proteoglycans Dermatan sulfate proteoglycans Heparin Heparan sulfate (syndecan) Hyaluronate – COO Function CH2OH O H H O Cell migration in: Embryogenesis Morphogenesis Wound healing Formation of bone, cartilage, cornea Transparency of cornea Transparency of cornea Binds LDL to plasma walls Anticoagulant (binds antithrombin III) Causes release of lipoprotein lipase from capillary walls Component of skin fibroblasts and aortic wall; commonly found on cell surfaces H H OH H H O H OH Glucuronic acid H – COO H H CH2OSO3 O HO H O OH H H H OH Glucuronic acid H Synovial lining Cartilage Calcified cartilage Synovial cavity Capsule NHCOCH3 N – Acetylgalactosamine – H O H COO – O CH2OSO3 O H H H O OH H H OH H H OSO3– H NHSO3– Glucuronic acid α (1 4) Glucosamine Keratan sulfate CH2OH O HO H H – CH2OSO3 O H O O H H OH H H OH H NHCOCH3 Galactose β (1 4) N – Acetylglucosamine Dermatan sulfate – H Bone H H H β (1 3) O Heparin H The functional properties of a normal joint depend, in part, on the presence of a soft, well-lubricated, deformable, and compressible layer of cartilaginous tissue covering the ends of the long bones that constitute the joint In Sis Lupus’ case, the pathologic process that characterizes SLE disrupted the structural and functional integrity of her articular (joint) cartilage NHCOCH3 N – Acetylglucosamine Chondroitin 6– sulfate – O the precursors that add sugar units, one at a time, first to the protein and then to the nonreducing end of the growing carbohydrate chain (Fig 49.10) Glycosylation occurs initially in the lumen of the ER and subsequently in the Golgi complex Glycosyltransferases, the enzymes that add sugars to the chain, are specific for the sugar being added, the type of linkage that is formed, and the sugars already present in the chain Once the initial sugars are attached to the protein, the alternating action of two glycosyltransferases adds the sugars of the repeating disaccharide to the growing glycosaminoglycan chain Sulfation occurs after addition of the sugar 3Ј-Phosphoadenosine 5Ј-phosphosulfate (PAPS), also called active sulfate, provides the sulfate groups (see Fig 33.34) An epimerase converts glucuronic acid residues to iduronic acid residues After synthesis, the proteoglycan is secreted from the cell Its structure resembles a bottle brush, with many glycosaminoglycan chains extending from the core protein (Fig 49.11) The proteoglycans may form large aggregates, noncovalently attached by a “link” protein to hyaluronic acid (Fig 49.12) The proteoglycans interact with the adhesion protein, fibronectin, which is attached to the cell membrane protein integrin Cross-linked fibers of collagen also associate with this complex, forming the ECM (Fig 49.13) H H HO β (1 3) O O H COO– O OH H H H OH Iduronic acid O3S CH2OH O O H H H H H β (1 3) O NHCOCH3 N – Acetylgalactosamine Fig 49.8 Repeating disaccharides of some glycosaminoglycans These repeating disaccharides usually contain an N-acetylated sugar and a uronic acid, which usually is glucuronic acid or iduronic acid Sulfate groups are often present but are not included in the sugar names in this figure 914 SECTION EIGHT / TISSUE METABOLISM Core protein Glycosaminoglycan B Link trisaccharide Galactose A Galactose O Xylose N H CH2 C H Serine O C n Uronic acid N –Acetylated sugar Fig 49.9 Attachment of glycosaminoglycans to proteins The sugars are linked to a serine or threonine residue of the protein A and B represent the sugars of the repeating disaccharide n B A PAP PAPS B A UDP UDP UDP UDP UDP A UDP UDP B A 6 A UDP Xyl–transferase Gal– transferase I Gal– transferase II GlcUA–transferase I GalNAc– transferase I GlcUA–transferase II GalNAc– transferase II Sulfotransferase UDP UDP UDP A UDP UDP A B B B UDP Protein core Xylose Galactose N – Acetylgalactosamine Glucuronic acid Sulfate Fig 49.10 Synthesis of chondroitin sulfate Sugars are added to the protein one at a time, with UDP-sugars serving as the precursors Initially a xylose residue is added to a serine in the protein Then two galactose residues are added, followed by a glucuronic acid (GlcUA) and an Nacetylglucosamine (GalNAc) Subsequent additions occur by the alternating action of two enzymes that produce the repeating disaccharide units One enzyme (6) adds GlcUA residues, and the other (7) adds GalNAc As the chain grows, sulfate groups are added by phosphoadenosine phosphosulfate (PAPS) Modified from Roden L In: Fishman WH, ed Metabolic Conjugation and Metabolic Hydrolysis, vol II Orlando, FL: Academic Press, 1970:401 915 CHAPTER 49 / THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE Table 49.4 Defective Enzymes in the Mucopolysaccharidoses Disease Hunter Hurler ϩ Scheie Maroteaux-Lamy Mucolipidosis VII Sanfilippo A Sanfilippo B Sanfilippo D Enzyme Deficiency – Accumulated Products Iduronate sulfatase ␣-L-Iduronidase N-Acetylgalactosamine sulfatase -Glucuronidase Heparan sulfamidase N-Acetylglucosaminidase N-Acetylglucosamine 6-sulfatase Heparan sulfate, Dermatan sulfate Heparan sulfate, Dermatan sulfate Dermatan sulfate Heparan sulfate, Dermatan sulfate Heparan sulfate Heparan sulfate Heparin sulfate – – – – n – – – – – – – – – – – – n – n – – – – – – n Core protein These disorders share many clinical features, although there are significant variations between disorders, and even within a single disorder, based on the amount of residual activity remaining In most cases, multiple organ systems are affected (with bone and cartilage being a primary target) For some disorders, there is significant neuronal involvement, leading to mental retardation – n – – – – Repeating disaccharide DEGRADATION OF PROTEOGLYCANS Lysosomal enzymes degrade proteoglycans, glycoproteins, and glycolipids, which are brought into the cell by the process of endocytosis Lysosomes fuse with the endocytic vesicles, and lysosomal proteases digest the protein component The carbohydrate component is degraded by lysosomal glycosidases Lysosomes contain both endoglycosidases and exoglycosidases The endoglycosidases cleave the chains into shorter oligosaccharides Then exoglycosidases, specific for each type of linkage, remove the sugar residues, one at a time, from the nonreducing ends Deficiencies of lysosomal glycosidases cause partially degraded carbohydrates from proteoglycans, glycoproteins, and glycolipids to accumulate within membrane-enclosed vesicles inside cells These “residual bodies” can cause marked enlargement of the organ with impairment of its function In the clinical disorder known as the mucopolysaccharidoses (caused by accumulation of partially degraded glycosaminoglycans), deformities of the skeleton may occur (Table 49.4) Mental retardation often accompanies these skeletal changes Fig 49.11 “Bottle-brush” structure of a proteoglycan, with a magnified segment Chondroitin sulfate Protein II INTEGRINS Integrins are the major cellular receptors for ECM proteins and provide a link between the internal cytoskeleton of cells (primarily the actin microfilament system) and extracellular proteins, such as fibronectin, collagen, and laminin Integrins Proteoglycan Fibronectin Collagen Link proteins Keratan sulfate Hyaluronic acid Fig 49.12 Proteoglycan aggregate Cell membrane Integrin Fig 49.13 Interactions between the cell membrane and the components of the extracellular matrix 916 SECTION EIGHT / TISSUE METABOLISM consist of an ␣ and a  subunit There are 18 distinct ␣ and eight distinct  gene products Twenty-four unique ␣/ dimers have been discovered Mice have been genetically engineered to be unable to express many of the integrin genes (one gene at a time), and the phenotypes of these knockout mice vary from embryonic lethality (the ␣5 gene is an example) to virtually no observable defects (as exemplified by the ␣1 gene) In addition to anchoring the cell’s cytoskeleton to the ECM, thereby providing a stable environment in which the cell can reside, the integrins are also involved in a wide variety of cell signaling options Certain integrins, such as those associated with white blood cells, are normally inactive because the white cell must circulate freely in the bloodstream However, if an infection occurs, cells located in the area of the infection release cytokines, which activate the integrins on the white blood cells, allowing them to bind to vascular endothelial cells (leukocyte adhesion) at the site of infection Leukocyte adhesion deficiency (LAD) is a genetic disorder that results from mutations in the 2 integrin such that leukocytes cannot be recruited to the sites of infection Conversely, drugs are now being developed to block either the 2 or ␣4 integrins (on lymphocytes) to treat inflammatory and autoimmune disorders by interfering with the normal white cell response to cytokines Integrins can be activated by “inside-out” mechanisms, whereby intracellular signaling events activate the molecule, or “outside-in” mechanisms, in which a binding event with the extracellular portion of the molecule initiates intracellular signaling events For those integrins that bind cells to ECM components, activation of specific integrins can result in migration of the affected cell through the ECM This mechanism is operative during growth, during cellular differentiation, and in the process of metastasis of malignant cells to neighboring tissues III ADHESION PROTEINS Fibronectin was first discovered as a large, external transformationsensitive protein (LETS), which was lost when fibroblasts were transformed into tumor cells Many tumor cells secrete less than normal amounts of adhesion protein material, which allows for more movement within the extracellular milieu This, in turn, increases the potential for the tumor cells to leave their original location and take root at another location within the body (metastasis) Because MMPs degrade extracellular matrix (ECM) components, their expression is important to allow cell migration and tissue remodeling during growth and differentiation In addition, many growth factors bind to ECM components and, as a bound component, not exhibit their normal growth-promoting activity Destruction of the ECM by the MMPs releases these growth factors, thereby allowing them to bind to cell surface receptors to initiate growth of tissues Thus, coordinated expression of the MMPs is required for appropriate cell movement and growth Cancer cells that metastasize require extensive ECM remodeling and usually use MMP activity to spread throughout the body Adhesion proteins are found in the ECM and link integrins to ECM components Adhesion proteins, of which fibronectin is a prime example, are large multidomain proteins that allow binding to many different components simultaneously In addition to integrin binding sites, fibronectin contains binding sites for collagen and glycosaminoglycans As the integrin molecule is bound to intracellular cytoskeletal proteins, the adhesion proteins provide a bridge between the actin cytoskeleton of the cell and the cells’ position within the ECM Loss of adhesion protein capability can lead to either physiologic or abnormal cell movement Alternative splicing of fibronectin allows many different forms of this adhesion protein to be expressed, including a soluble form (versus cell-associated forms), which is found in the plasma The metabolic significance of these products remains to be determined IV MATRIX METALLOPROTEINASES The ECM contains a series of proteases known as the matrix metalloproteinases, or MMPs These are zinc-containing proteases that use the zinc to appropriately position water to participate in the proteolytic reaction At least 23 different types of human MMPs exist, and they cleave all proteins found in the ECM, including collagen and laminin A propeptide is present in newly synthesized MMPs that contains a critical cysteine residue The cysteine residue in the propeptide binds to the zinc atom at the active site of the protease and prevents the propeptide from exhibiting proteolytic activity Removal of the propeptide is required to activate the MMPs Once activated, certain MMPs can activate other forms of MMP Regulation of MMP activity is quite complex These regulatory processes include transcriptional regulation, proteolytic activation, inhibition by the circulating protein ␣2-macroglobulin, and regulation by a class of inhibitors known as CHAPTER 49 / THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE 917 tissue inhibitors of metalloproteinases, or TIMPs It is important that the synthesis of TIMPs and MMPs be coordinately regulated, because dissociation of their expression can facilitate various clinical disorders, such as certain forms of cancer and atherosclerosis CLINICAL COMMENTS Articular cartilage is a living tissue with a turnover time determined by a balance between the rate of its synthesis and that of its degradation (Fig 49.14) The chondrocytes that are embedded in the matrix of intraarticular cartilage participate in both its synthesis and its enzymatic degradation The latter occurs as a result of cleavage of proteoglycan aggregates by enzymes produced and secreted by the chondrocytes In SLE, the condition that affects Sis Lupus, this delicate balance is disrupted in favor of enzymatic degradation, leading to dissolution of articular cartilage and, with it, the loss of its critical cushioning functions The underlying mechanisms responsible for this process in SLE include the production of antibodies directed against specific cellular proteins in cartilage as well as in other intra-articular tissues The cellular proteins thus serve as the “antigens” to which these antibodies react In this sense, SLE is an “autoimmune” disease because antibodies are produced by the host that attack “self” proteins This process excites the local release of cytokines such as interleukin-1 (IL-1), which increases the proteolytic activity of the chondrocytes, causing further loss of articular proteins such as the proteoglycans The associated inflammatory cascade is responsible for Sis Lupus’ joint pain Pericellular matrix Intercellular matrix Synovial fluid Chondrocyte Biosynthesis t1 = 100– 800 d t = – 30 d Degradation products Lysosomal 0degradation Fig 49.14 Synthesis and degradation of proteoglycans by chondrocytes From Cohen RD, et al The Metabolic Basis of Acquired Disease, vol London: Bailliere Tindall, 1990:1859 918 SECTION EIGHT / TISSUE METABOLISM The microvascular complications of both type and type diabetes mellitus involve the small vessels of the retina (diabetic retinopathy), the renal glomerular capillaries (diabetic nephropathy), and the vessels supplying blood to the peripheral nerves (autonomic neuropathy) The lack of adequate control of Ann Sulin’s diabetic state over many years caused a progressive loss of the filtering function of the approximately one-and-one-half million glomerular capillary–mesangial units that are present in her kidneys Chronic hyperglycemia is postulated to be a major metabolic initiator or inducer of diabetic microvascular disease, including those renal glomerular changes that often lead to end-stage renal disease (“glucose toxicity”) For a comprehensive review of the four postulated molecular mechanisms by which chronic hyperglycemia causes these vascular derangements, the reader is referred to an excellent review by Sheetz and King (see suggested references) Regardless of which of the postulated mechanisms (increased flux through the aldose reductase or polyol pathway [see Chapter 30], the generation of advanced glycosylation end products [AGEs], the generation of reactive oxygen intermediates [see Chapter 24], or excessive activation of protein kinase C [see Chapter 18]) will eventually be shown to be the predominant causative mechanism, each can lead to the production of critical intracellular and extracellular signaling molecules (e.g., cytokines) These, in turn, can cause pathologic changes within the glomerular filtration apparatus that reduce renal function These changes include: (1) increased synthesis of collagen, type IV, fibronectin, and some of the proteoglycans, causing the glomerular basement membrane (GBM; Fig 49.15) to become diffusely thickened throughout the glomerular capillary network This membrane thickening alters certain specific filtration properties of the GBM, preventing some of the metabolites that normally enter the urine from the glomerular capillary blood (via the fenestrated capillary endothelium) from doing so (a decline in glomerular filtration rate or GFR) As a result, these potentially toxic substances accumulate in the blood and contribute to the overall clinical presentation of advancing uremia In spite of the Glomerulus Capillary loops Fenestrated capillary endothelium Urinary space Capillary lumen Capillary lumen Parietal epithelium (Bowmans capsule) Proximal tubule Bowmans space (urinary space) Urine Mesangial cells Mesangial matrix Glomerular basement membrane Bowmans space (urinary space) (proximal-most part of a nephron) Capillary lumen Capillary lumen Fig 49.15 A cross-section of a normal renal glomerulus showing four capillary tufts delivering blood to the glomerulus for filtration across the fenestrated capillary endothelium then through the glomerular basement membrane into the Bowman’s space to form urine The urine then enters the proximal tubule of the nephron This filtration removes potentially toxic metabolic end products from the blood The mesangium, by contracting and expanding, controls the efficiency of these filtering and excretory functions by regulating the hydraulic filtration pressures within the glomerulus An intact basement membrane must be present to maintain the integrity of the filtering process CHAPTER 49 / THE EXTRACELLULAR MATRIX AND CONNECTIVE TISSUE thickening of the GBM, this membrane becomes “leaky” for some macromolecules (e.g., albumin) that normally not enter the urine from the glomerular capillaries (microalbuminuria) Suggested mechanisms for this increased permeability or leakiness include reduced synthesis of the specific proteoglycan, heparan sulphate, as well as increased basement membrane production of vascular endothelium growth factor (VEGF), a known angiogenic and permeability factor; and expansion of the extracellular matrix in the mesangium The mesangium consists of specialized tissue containing collagen, proteoglycans, and other macromolecules that surround the glomerular capillaries and that, through its gel-like and sieving properties, determine, in part, the glomerular capillary hydraulic filtration pressure as well as the functional status of the capillary endothelium–mesangial glomerular basement membrane filtration apparatus (see Fig 49.15) As the mesangial tissue expands, the efficiency of glomerular filtration diminishes proportionately The cause of these mesangial changes is, in part, the consequence of increased expression of certain growth factors, especially transforming growth factor  (TGF-) and connective tissue growth factor (CTGF) Current therapeutic approaches in patients with early diabetic nephropathy include the use of antibodies that neutralize TGF- BIOCHEMICAL COMMENTS Osteogenesis imperfecta (OI) is a heterogenous group of diseases that have in common a defect in collagen production This defect can be either of two types: The first type is associated with a reduction in the synthesis of normal collagen (due to a gene deletion or splice-site mutation) The second type is associated with the synthesis of a mutated form of collagen Most of the mutations have a dominant-negative effect, leading to an autosomal dominant mode of transmission In the second type of OI, many of the known mutations involve substitutions of another amino acid for glycine This results in an unstable collagen molecule, because glycine is the only amino acid that can fit between the other two chains within the triple helix of collagen If the mutation is near the carboxy-terminal of the molecule, the phenotype of the disease is usually more severe than if the mutation is near the aminoterminal end (recall that triple helix formation proceeds from the carboxy- to the aminoterminal end of the molecule) Of interest are mutations that replace glycine with either serine or cysteine Such mutations are more stable than expected, because of the hydrogen-bonding capabilities of serine and the ability of cysteine to form disulfide bonds Both would aid in preventing the strands of the triple helix from unwinding Children with OI can be treated with a class of compounds known as bisphosphonates, which consist of two phosphates linked by a carbon or nitrogen bridge (thus, they are analogs of pyrophosphate, in which the two phosphates are linked by oxygen) Normal bone remodeling is the result of a coordinated “coupling” between osteoclast activity (cells that resorb bone) and osteoblast activity (cells that form bone) In OI, bone resorption outpaces bone formation because osteoclast activity is enhanced (perhaps because of the reduced levels of normal collagen present to act as nucleating sites for bone formation) This leads to a net loss of bone mass and fragility of the skeleton Bisphosphonates inhibit osteoclast action with the potential to increase bone mass and its tensile strength Suggested References Bosman FT, Stamenhovic I Functional structure and composition of the extracellular matrix J Pathol 2003;200:423–428 Byers PH Disorders of collagen biosynthesis and structure In: Scriver CR, Beaudet AL, Valle D, Sly WS, et al., eds The Metabolic and Molecular Bases of Inherited Disease, vol IV, 8th Ed New York: McGraw-Hill, 2001:5241–5286 919 920 SECTION EIGHT / TISSUE METABOLISM Hynes RO Integrins: biodirectional allosteric signalling machines Cell 2002;110:673–687 Neufeld EF, Muenzer J The mucopolysaccharidoses In: Scriver CR, Beaudet AL, Valle D, Sly WS, et al., eds The Metabolic and Molecular Bases of Inherited Disease, vol IV, 8th Ed New York: McGraw-Hill, 2001:3421–3452 Sheetz MJ, King GL Molecular understanding of hyperglycemia’s adverse effects for diabetic complications JAMA 2002;288(20):2579–2588 REVIEW QUESTIONS—CHAPTER 49 Individuals who develop scurvy suffer from sore and bleeding gums and loss of teeth This is due, in part, to the synthesis of a defective collagen molecule The step that is affected in collagen biosynthesis attributable to scurvy is which of the following? (A) The formation of disulfide bonds, which initiates tropocollagen formation (B) The formation of lysyl cross-links between collagen molecules (C) Secretion of tropocollagen into the extracellular matrix (D) The formation of collagen fibrils (E) The hydroxylation of proline residues, which stabilizes the collagen structure The underlying mechanism that allows elastin to exhibit elastic properties (expand and contract) is which of the following? (A) Proteolysis during expansion, and resynthesis during contraction (B) Breaking of disulfide bonds during expansion, reformation of these bonds during contraction (C) A decrease in entropy during expansion, and an increase in entropy during contraction (D) The breaking of salt bridges during expansion, and reformation of the salt bridges during contraction (E) Hydroxylation of elastin during expansion, and decarboxylation of elastin during contraction The underlying mechanism by which glycosaminoglycans allow for the formation of a gel-like substance in the extracellular matrix in which of the following? (A) Charge attraction between glycosaminoglycan chains (B) Charge repulsion between glycosaminoglycan chains (C) Hydrogen bonding between glycosaminoglycan chains (D) Covalent cross-linking between glycosaminoglycan chains (E) Hydroxylation of adjacent glycosaminoglycan chains The movement of tumor cells from their site of origin to other locations within the body requires the activity of which of the following proteins? (A) Collagen (B) Laminin (C) Proteoglycans (D) Elastin (E) Matrix metalloproteinases Fibronectin is frequently absent in malignant fibroblast cells One of the major functions of fibronectin is which of the following? (A) To inhibit the action of matrix metalloproteinases (B) To coordinate collagen deposition within the extracellular matrix (C) To fix the position of cells within the extracellular matrix (D) To regulate glycosaminoglycan production (E) To extend glycosaminoglycan chains using nucleotide sugars ... Mitochondrial damage Membrane damage SER RER DNA damage Nucleus (DNA) DNA O 2 OH• H2O Na+ Ca Cell swelling 2+ Increased permeability Massive influx of Ca2+ Lipid peroxidation Fig 24 .7 Free radical–mediated... Vitamin C Vitamin E O2Ϫ ϩ eϪ ϩ 2Hϩ yields H2O2 O2Ϫ ϩ 2Hϩ yields H2O2 ϩ O2 O2Ϫ ϩ HO•ϩ Hϩ yields CO2 ϩ H2O H2O2 ϩ O2 yields H2O O2Ϫ ϩ H2O2 ϩ Hϩ yields H2O ϩ O2 The mechanism of vitamin E as an antioxidant... damage increases VLDL and protein accumulation (6) Cell damage leads to release of the hepatic enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) oxidation is decreased