Cell injury - etiology and pathogenetic mechanisms

Cells normally live in homeostasis, a steady internal state. When a stress exceeds the cell's ability to adapt, the cell becomes injured. The points below carry the whole topic.

• The most common cause of cell injury is hypoxia (oxygen deficiency), often produced by ischaemia. Chemical, physical, infective, immunological, nutritional, genetic and age-related causes also injure cells.
• Injury works through a small number of mechanisms that usually act together: ATP depletion, mitochondrial damage, disturbed calcium homeostasis and free radical generation.
• When ATP falls, the Na⁺/K⁺ pump fails. Na⁺, Ca²⁺ and water enter the cell and it swells. Anaerobic glycolysis lowers the pH.
• Raised cytosolic Ca²⁺ activates phospholipases, proteases, endonucleases and ATPases, which damage membranes, the cytoskeleton and DNA.
• Reactive oxygen species (superoxide, hydrogen peroxide, hydroxyl radical) damage lipids, proteins and DNA. Lipid peroxidation is a chain reaction that continues until an antioxidant stops it.
• The antioxidant system is enzymatic (superoxide dismutase, catalase, glutathione peroxidase) and non-enzymatic (vitamins A, C and E, glutathione and others).
• Restoring blood flow after ischaemia is essential, but reperfusion can cause injury of its own through ROS, complement and leukocytes.

Homeostasis is the maintenance of a constant internal environment in the body by co-ordinated physiological mechanisms. Cells exist in homeostasis. When they are subjected to stress they attempt to adapt. If they cannot adapt, they become injured cells. Adaptation is covered in topic 002.

Injury is reversible or irreversible. Mild injury is reversible: the cell returns to homeostasis when the stimulus is removed. Severe or progressive injury is irreversible and ends in cell death, by necrosis or apoptosis (topic 003).

The lecture figure below compares a normal cell with an injured one. The injured cell shows blebs in the plasma membrane, swollen and distorted mitochondria and endoplasmic reticulum (ER), ribosomes lost from the ER surface, vacuolated cytoplasm and clumping of nuclear chromatin.

The most common cause is hypoxia, which prevents aerobic oxidative respiration and the conversion of carbohydrates into ATP. Hypoxia is often caused by ischaemia (lack of blood supply to an area), but also by pneumonia (less oxygen reaching the blood), anaemia (less oxygen-carrying capacity) and carbon monoxide poisoning.

Other causes, as set out in the lecture figure below, are direct contact with harmful agents, pressure, hypothermia, hyperthermia, trauma, electric current, solar and ionising radiation, infection and immune defects.

Radiation injures cells in two ways. The direct effect is energy absorbed straight into DNA, which breaks the strands. The indirect effect is that radiation ionises water, and the products, including hydroxyl radicals and hydrated electrons, damage DNA. The figure below shows both routes, followed by DNA repair, cell-cycle checkpoint control and survival or death of the cell.

A free radical is a molecule with an unpaired electron in its outer shell. This makes it unstable and highly reactive with molecules nearby. Reactive oxygen species (ROS) are oxygen-containing molecules, including free radicals, that react with biological structures. The main ones are:

• superoxide anion (O₂•⁻)
• hydrogen peroxide (H₂O₂), which is a ROS but not a free radical
• hydroxyl radical (•OH), the most damaging
• nitric oxide (NO) and its product peroxynitrite (ONOO⁻)
• hypochlorous acid (HOCl)
• alkoxy radicals (RO•) and peroxyl radicals (ROO•)

Free radicals are generated under normal physiological conditions, and the body normally deals with them through antioxidants. Injury follows when pathological processes produce high levels of free radicals that the body cannot cope with.

Sources. Free radicals form normally in the body, mainly in aerobic respiration (incomplete reduction of O₂ in the mitochondria). They are also made by NADPH oxidase in membranes and cytoplasm, by cytochrome P450 in the ER, in peroxisomes, and during the metabolic burst of phagocytosis. Inflammation and detoxification of chemicals produce them too. Ionising and ultraviolet radiation, air pollutants, smoking and reperfusion add to the load.

The lecture figure below shows the main sources and the balance between harmful and useful effects.

Oxidative stress is the state in which ROS generation exceeds the body's ability to neutralise and eliminate them. It leads to oxidation of cell components, activation of signal transduction pathways, DNA damage and changes in gene expression.

The Fenton reaction is the key source of the hydroxyl radical. Iron (Fe²⁺), or copper, reacts with hydrogen peroxide to give •OH. The mitochondrial picture below shows the sequence from superoxide to hydroxyl radical, and the pathological effects.

Free radicals harm cells in three main ways.

• Lipid peroxidation. Hydroxyl radicals attack the lipids of cell membranes. Each attack produces a new radical, so the damage spreads in a chain reaction that destroys membranes and organelles.
• Protein oxidation. Oxidised proteins cross-link, misfold and lose enzyme activity.
• DNA damage. Oxidation, fragmentation and mutation of DNA. This can contribute to oncogenesis.

The figure below shows the chain reaction. Radicals are produced by ionising radiation or an exogenous chemical, and each step generates a new radical R• that damages critical constituents X, Y and Z. The cell's antioxidants (AO) and scavenger molecules (S) interact with the radicals to form stable products, which stops the chain.

One clinical example is carbon tetrachloride, which is converted into the trichloromethyl radical. This causes liver swelling, decreased protein synthesis and, through hepatocyte dysfunction, fatty liver. Iron overload works the same way: it generates free radicals through the Fenton reaction, which is the tissue-damaging mechanism in haemochromatosis.

Membrane damage. Several pathways converge on the plasma membrane. Oxygen deficiency lowers ATP and so reduces phospholipid resynthesis. ROS cause lipid peroxidation. Raised cytosolic Ca²⁺ activates phospholipase, which breaks down phospholipids, and activates proteases, which damage the cytoskeleton. Together these produce membrane damage.

Antioxidants are natural and synthetic molecules that inhibit the reactions of ROS with biological structures, or prevent their uncontrolled formation. They are either enzymatic or non-enzymatic.

The lecture figure summarises the enzymatic and non-enzymatic system.

ATP is made by oxidative phosphorylation in mitochondria (aerobic) and by glycolysis (anaerobic). In hypoxia or ischaemia, mitochondria cannot oxidatively phosphorylate, so ATP falls. This is the most common mechanism of cell injury, and it works on several fronts.

• Failure of the Na⁺/K⁺ pump. Na⁺ and water enter the cell and K⁺ leaves. The ER dilates, the cell swells, microvilli are lost and blebs form. The falling intracellular K⁺ depolarises the membrane, allowing Cl⁻ to enter and the cell to swell further.
• Anaerobic glycolysis. Glycogen stores fall, lactic acid accumulates and pH falls. The acidity damages enzymes and clumps nuclear chromatin.
• Ribosome detachment. Ribosomes detach from the ER, so protein synthesis falls.
• Lipid deposition. Failed lipoprotein export leads to fat accumulation.

The changes are reversible if oxygen is restored. Further depletion of oxygen leads to necrosis.

Ca²⁺ is a second messenger, so the cell keeps its intracellular concentration very low, far below the extracellular level. A Ca²⁺/Mg²⁺ ATPase and exchange systems pump Ca²⁺ out, and most intracellular Ca²⁺ is stored in mitochondria and the ER.

In injury, Ca²⁺ enters the cell from outside and is released from the ER and mitochondria. Raised cytosolic Ca²⁺ activates:

• phospholipase, which breaks down phospholipids and damages membranes
• protease, which damages membranes and cytoskeletal filaments
• endonuclease, which damages the nucleus
• ATPase, which uses up ATP

Mitochondrial permeability also rises. The figure shows this whole pathway in a single epithelial cell.

Mitochondria are central to cell injury. Their damage causes ATP depletion, high Ca²⁺ and ROS, which are the common causes of injury. A mitochondrial permeability transition pore forms. Its opening increases the membrane's conductance and abolishes the membrane potential, so oxidative phosphorylation fails. The result is a fall in ATP and then necrosis.

Mitochondria also release cytochrome c during apoptosis, which is covered in topic 003. The lecture figure below shows the swollen mitochondria with amorphous densities that mark the progression of injury.

Ischaemia-reperfusion (I-R) injury is damage caused when blood flow is restored to an ischaemic organ. Restoring flow is essential to prevent irreversible cell injury, but reperfusion can augment the injury.

Clinical settings. Ischaemia occurs in myocardial infarction, peripheral vascular insufficiency, stroke and hypovolaemic shock. Reperfusion follows thrombolytic therapy, organ transplantation, coronary angioplasty, aortic cross-clamping and cardiopulmonary bypass. Severe cases produce a systemic inflammatory response syndrome (SIRS) or multiple organ dysfunction syndrome (MODS). These account for 30 to 40 per cent of mortality in a tertiary referral ICU.

Cellular changes during ischaemia. These are altered membrane potential, altered ion distribution (raised intracellular Ca²⁺ and Na⁺), cell swelling, cytoskeletal disorganisation, increased hypoxanthine, decreased ATP, decreased phosphocreatine and cellular acidosis.

Endothelium. Ischaemic endothelium expresses proinflammatory gene products, such as leukocyte adhesion molecules and cytokines, and bioactive agents such as endothelin and thromboxane A₂. It represses protective products, such as constitutive nitric oxide synthase, thrombomodulin, prostacyclin and nitric oxide.

Reactive oxygen species. These include O₂•⁻, •OH, HOCl, H₂O₂ and peroxynitrite. They damage membranes by lipid peroxidation. They activate membrane phospholipase A₂ to form arachidonic acid, which gives thromboxane A₂ and leukotriene B₄, and they increase leukocyte activation, chemotaxis and adherence to endothelium.

Complement. I-R activates complement, forming C3a, C5a, iC3b and C5b-9. The most potent is C5a. Complement compromises blood flow by altering vascular homeostasis and increasing leukocyte adherence.

Leukocytes. I-R activates leukocytes, which adhere to endothelium and migrate across it. They cause mechanical obstruction, and release toxic ROS, proteases and elastases. These increase microvascular permeability, oedema and thrombosis, and kill parenchymal cells.

Manifestations. These include vascular injury and the no-reflow phenomenon, myocardial stunning, reperfusion arrhythmias (ventricular tachycardia, ventricular fibrillation and idioventricular rhythms), CNS and GI I-R injury, and multiple organ dysfunction. Risk factors include hypercholesterolaemia, hypertension and diabetes.

Prevention and treatment. Strategies in the lecture are:

• Ischaemic preconditioning. Brief periods of ischaemia protect against prolonged I-R injury. It raises cellular adenosine and protects energy stores or inhibits leukocyte adherence. It has been used in coronary artery bypass grafting and before hepatic resection.
• Antioxidant therapy. Superoxide dismutase, catalase, mannitol, allopurinol, vitamin E, N-acetylcysteine, iron chelators, ACE inhibitors and calcium channel antagonists have been tried. Human recombinant superoxide dismutase was tried in patients with haemorrhagic shock, and SOD in cadaveric renal transplantation. The lecture calls the results equivocal.
• Anticomplement therapy. A C3 convertase inhibitor, soluble complement receptor 1 (which reduced infarct size by 44 per cent in a rat model of myocardial I-R), and a humanised anti-C5 antibody fragment (h5G1.1-scFv).
• Antileukocyte therapy. Leukocyte depletion or filtration, soluble interleukin-1 receptor antagonists, anti-tumour necrosis factor antibodies, platelet-activating factor and leukotriene B₄ antagonists, and aspirin-triggered lipoxins that block neutrophil chemotaxis, adhesion and migration.
• Controlled, graded reperfusion.

The lecture concludes that timely reperfusion remains the cornerstone of practice. Inhibiting inflammation may disrupt protective responses or cause immunosuppression.