Atherosclerosis

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Atherosclerosis
Synonyms	Arteriosclerotic vascular disease (ASVD)
The progression of atherosclerosis (narrowing exaggerated)
Specialty	Cardiology, angiology
Symptoms	None[1]
Complications	Coronary artery disease, stroke, peripheral artery disease, kidney problems[1]
Usual onset	Youth (worsens with age)[2]
Causes	Unknown[1]
Risk factors	High blood pressure, diabetes, smoking, obesity, family history, unhealthy diet[3]
Prevention	Healthy diet, exercise, not smoking, maintaining a normal weight[4]
Medication	Statins, high blood pressure medication, aspirin[5]
Frequency	~100% (>65 years old)[6]

Atherosclerosis is a disease in which the inside of an artery narrows due to the build up of plaque. Initially, there are generally no symptoms. When severe, it can result in coronary artery disease, stroke, peripheral artery disease, or kidney problems, depending on which arteries are affected. Symptoms, if they occur, generally do not begin until middle age.

The exact cause is not known.^[1] Risk factors include abnormal cholesterol levels, high blood pressure, diabetes, smoking, obesity, family history, and an unhealthy diet.^[3] Plaque is made up of fat, cholesterol, calcium, and other substances found in the blood.^[7] The narrowing of arteries limits the flow of oxygen-rich blood to parts of the body.^[7] Diagnosis is based upon a physical exam, electrocardiogram, and exercise stress test, among others.^[8]

Prevention is generally by eating a healthy diet, exercising, not smoking, and maintaining a normal weight.^[4] Treatment of established disease may include medications to lower cholesterol such as statins, blood pressure medication, or medications that decrease clotting, such as aspirin.^[5] A number of procedures may also be carried out such as percutaneous coronary intervention, coronary artery bypass graft, or carotid endarterectomy.^[5]

Atherosclerosis generally starts when a person is young and worsens with age.^[2] Almost all people are affected to some degree by the age of 65.^[6] Atherosclerosis is the number one cause of death and disability in the developed world.^[9] Atherosclerosis was first described in 1575.^[10] There is evidence, however, that the condition occurred in people more than 5,000 years ago.^[10]

Definitions

The following terms are similar, yet distinct, in both spelling and meaning, and can be easily confused: arteriosclerosis, arteriolosclerosis, and atherosclerosis. Arteriosclerosis is a general term describing any hardening (and loss of elasticity) of medium or large arteries (from Greek ἀρτηρία (artēria), meaning 'artery', and σκλήρωσις (sklerosis), meaning 'hardening'); arteriolosclerosis is any hardening (and loss of elasticity) of arterioles (small arteries); atherosclerosis is a hardening of an artery specifically due to an atheromatous plaque. The term atherogenic is used for substances or processes that cause formation of atheroma.^[11]

Signs and symptoms

Atherosclerosis is asymptomatic for decades because the arteries enlarge at all plaque locations, thus there is no effect on blood flow.^[12] Even most plaque ruptures do not produce symptoms until enough narrowing or closure of an artery, due to clots, occurs. Signs and symptoms only occur after severe narrowing or closure impedes blood flow to different organs enough to induce symptoms.^[13] Most of the time, patients realize that they have the disease only when they experience other cardiovascular disorders such as stroke or heart attack. These symptoms, however, still vary depending on which artery or organ is affected.^[14]

Typically, atherosclerosis begins in childhood, as a thin layer of white-yellowish streaks with the inner layers of the artery walls (an accumulation of white blood cells, mostly monocytes/macrophages) and progresses from there.

Clinically, given enlargement of the arteries for decades, symptomatic atherosclerosis is typically associated with men in their 40s and women in their 50s to 60s. Sub-clinically, the disease begins to appear in childhood, and rarely is already present at birth. Noticeable signs can begin developing at puberty. Though symptoms are rarely exhibited in children, early screening of children for cardiovascular diseases could be beneficial to both the child and his/her relatives.^[15] While coronary artery disease is more prevalent in men than women, atherosclerosis of the cerebral arteries and strokes equally affect both sexes.^[16]

Marked narrowing in the coronary arteries, which are responsible for bringing oxygenated blood to the heart, can produce symptoms such as the chest pain of angina and shortness of breath, sweating, nausea, dizziness or light-headedness, breathlessness or palpitations.^[14] Abnormal heart rhythms called arrhythmias (the heart is either beating too slow or too fast) are another consequence of ischemia.^[17]

Carotid arteries supply blood to the brain and neck.^[17] Marked narrowing of the carotid arteries can present with symptoms such as a feeling of weakness, not being able to think straight, difficulty speaking, becoming dizzy and difficulty in walking or standing up straight, blurred vision, numbness of the face, arms, and legs, severe headache and losing consciousness. These symptoms are also related to stroke (death of brain cells). Stroke is caused by marked narrowing or closure of arteries going to the brain; lack of adequate blood supply leads to the death of the cells of the affected tissue.^[18]

Peripheral arteries, which supply blood to the legs, arms, and pelvis, also experience marked narrowing due to plaque rupture and clots. Symptoms for the marked narrowing are numbness within the arms or legs, as well as pain. Another significant location for the plaque formation is the renal arteries, which supply blood to the kidneys. Plaque occurrence and accumulation leads to decreased kidney blood flow and chronic kidney disease, which, like all other areas, are typically asymptomatic until late stages.^[14]

According to United States data for 2004, in about 66% of men and 47% of women, the first symptom of atherosclerotic cardiovascular disease is a heart attack or sudden cardiac death (death within one hour of onset of the symptom). Cardiac stress testing, traditionally the most commonly performed non-invasive testing method for blood flow limitations, in general, detects only lumen narrowing of ≈75% or greater, although some physicians claim that nuclear stress methods can detect as little as 50%.

Case studies have included autopsies of U.S. soldiers killed in World War II and the Korean War. A much-cited report involved autopsies of 300 U.S. soldiers killed in Korea. Although the average age of the men was 22.1 years, 77.3 percent had "gross evidence of coronary arteriosclerosis".^[19] Other studies done of soldiers in the Vietnam War showed similar results, although often worse than the ones from the earlier wars. Theories include high rates of tobacco use and (in the case of the Vietnam soldiers) the advent of processed foods after World War II.

Risk factors

Atherosclerosis and lipoproteins

See also: Lipoprotein and Lipoprotein (a)

The atherosclerotic process is not fully understood. Atherosclerosis is initiated by inflammatory processes in the endothelial cells of the vessel wall associated with retained low-density lipoprotein (LDL) particles.^[20] This retention may be a cause, an effect, or both, of the underlying inflammatory process.^[21]

The presence of the plaque induces the muscle cells of the blood vessel to stretch, compensating for the additional bulk, and the endothelial lining thickens, increasing the separation between the plaque and lumen. This somewhat offsets the narrowing caused by the growth of the plaque, but it causes the wall to stiffen and become less compliant to stretching with each heart beat.^[22]

Modifiable

• Diabetes^[23]
• Dyslipidemia^[23]
• Tobacco smoking^[23]
• Trans fat^[23]
• Abdominal obesity^[23]
• Western pattern diet^[23]
• Insulin resistance^[23]
• Hypertension^[23]

Nonmodifiable

• Advanced age^[23]
• Male^[23]
• Family history^[23]
• Genetic abnormalities^[23]

Lesser or uncertain

• South Asian descent^[24][25]
• Thrombophilia^[26][27][28]
• Saturated fat^[23][29]
• Excessive carbohydrates^[23][30]
• Elevated triglycerides^[23]
• Systemic inflammation^[31]
• Hyperinsulinemia^[32]
• Sleep deprivation^[33]
• Air pollution^[34][35]
• Sedentary lifestyle^[23]
• Arsenic poisoning^[36]
• Alcohol^[23]
• Chronic stress^[23]
• Hypothyroidism^[37]
• Periodontal disease^[38]

Dietary

The relation between dietary fat and atherosclerosis is controversial. Writing in Science, Gary Taubes detailed that political considerations played into the recommendations of government bodies.^[39] The USDA, in its food pyramid, promotes a diet of about 64% carbohydrates from total calories. The American Heart Association, the American Diabetes Association and the National Cholesterol Education Program make similar recommendations. In contrast, Prof Walter Willett (Harvard School of Public Health, PI of the second Nurses' Health Study) recommends much higher levels of fat, especially of monounsaturated and polyunsaturated fat.^[40] These dietary recommendations reach a consensus, though, against consumption of trans fats.

The role of dietary oxidized fats/lipid peroxidation (rancid fats) in humans is not clear. Laboratory animals fed rancid fats develop atherosclerosis. Rats fed DHA-containing oils experienced marked disruptions to their antioxidant systems, and accumulated significant amounts of phospholipid hydroperoxide in their blood, livers and kidneys.^[41]

Rabbits fed atherogenic diets containing various oils were found to undergo the greatest amount of oxidative susceptibility of LDL via polyunsaturated oils.^[42] In another study, rabbits fed heated soybean oil "grossly induced atherosclerosis and marked liver damage were histologically and clinically demonstrated."^[43] However, Fred Kummerow claims that it is not dietary cholesterol, but oxysterols, or oxidized cholesterols, from fried foods and smoking, that are the culprit.^[44]

Rancid fats and oils taste very bad even in small amounts, so people avoid eating them.^[45] It is very difficult to measure or estimate the actual human consumption of these substances.^[46] Highly unsaturated omega-3 rich oils such as fish oil are being sold in pill form so that the taste of oxidized or rancid fat is not apparent. The health food industry's dietary supplements are self-regulated and outside of FDA regulations.^[47] To properly protect unsaturated fats from oxidation, it is best to keep them cool and in oxygen-free environments.

Mechanism

Atherogenesis is the developmental process of atheromatous plaques. It is characterized by a remodeling of arteries leading to subendothelial accumulation of fatty substances called plaques. The buildup of an atheromatous plaque is a slow process, developed over a period of several years through a complex series of cellular events occurring within the arterial wall and in response to a variety of local vascular circulating factors. One recent hypothesis suggests that, for unknown reasons, leukocytes, such as monocytes or basophils, begin to attack the endothelium of the artery lumen in cardiac muscle. The ensuing inflammation leads to formation of atheromatous plaques in the arterial tunica intima, a region of the vessel wall located between the endothelium and the tunica media. The bulk of these lesions is made of excess fat, collagen, and elastin. At first, as the plaques grow, only wall thickening occurs without any narrowing. Stenosis is a late event, which may never occur and is often the result of repeated plaque rupture and healing responses, not just the atherosclerotic process by itself.

Cellular

Micrograph of an artery that supplies the heart showing significant atherosclerosis and marked luminal narrowing. Tissue has been stained using Masson's trichrome.

Early atherogenesis is characterized by the adherence of blood circulating monocytes (a type of white blood cell) to the vascular bed lining, the endothelium, then by their migration to the sub-endothelial space, and further activation into monocyte-derived macrophages.^[48] The primary documented driver of this process is oxidized lipoprotein particles within the wall, beneath the endothelial cells, though upper normal or elevated concentrations of blood glucose also plays a major role and not all factors are fully understood. Fatty streaks may appear and disappear.

Low-density lipoprotein (LDL) particles in blood plasma invade the endothelium and become oxidized, creating risk of cardiovascular disease. A complex set of biochemical reactions regulates the oxidation of LDL, involving enzymes (such as Lp-LpA2) and free radicals in the endothelium.

Initial damage to the endothelium results in an inflammatory response. Monocytes enter the artery wall from the bloodstream, with platelets adhering to the area of insult. This may be promoted by redox signaling induction of factors such as VCAM-1, which recruit circulating monocytes, and M-CSF, which is selectively required for the differentiation of monocytes to macrophages. The monocytes differentiate into macrophages, which proliferate locally,^[49] ingest oxidized LDL, slowly turning into large "foam cells" – so-called because of their changed appearance resulting from the numerous internal cytoplasmic vesicles and resulting high lipid content. Under the microscope, the lesion now appears as a fatty streak. Foam cells eventually die and further propagate the inflammatory process.

In addition to these cellular activities, there is also smooth muscle proliferation and migration from the tunica media into the intima in response to cytokines secreted by damaged endothelial cells. This causes the formation of a fibrous capsule covering the fatty streak. Intact endothelium can prevent this smooth muscle proliferation by releasing nitric oxide.

Calcification and lipids

Calcification forms among vascular smooth muscle cells of the surrounding muscular layer, specifically in the muscle cells adjacent to atheromas and on the surface of atheroma plaques and tissue.^[50] In time, as cells die, this leads to extracellular calcium deposits between the muscular wall and outer portion of the atheromatous plaques. With the atheromatous plaque interfering with the regulation of the calcium deposition, it accumulates and crystallizes. A similar form of an intramural calcification, presenting the picture of an early phase of arteriosclerosis, appears to be induced by a number of drugs that have an antiproliferative mechanism of action (Rainer Liedtke 2008).

Cholesterol is delivered into the vessel wall by cholesterol-containing low-density lipoprotein (LDL) particles. To attract and stimulate macrophages, the cholesterol must be released from the LDL particles and oxidized, a key step in the ongoing inflammatory process. The process is worsened if there is insufficient high-density lipoprotein (HDL), the lipoprotein particle that removes cholesterol from tissues and carries it back to the liver.

The foam cells and platelets encourage the migration and proliferation of smooth muscle cells, which in turn ingest lipids, become replaced by collagen and transform into foam cells themselves. A protective fibrous cap normally forms between the fatty deposits and the artery lining (the intima).

These capped fatty deposits (now called 'atheromas') produce enzymes that cause the artery to enlarge over time. As long as the artery enlarges sufficiently to compensate for the extra thickness of the atheroma, then no narrowing ("stenosis") of the opening ("lumen") occurs. The artery becomes expanded with an egg-shaped cross-section, still with a circular opening. If the enlargement is beyond proportion to the atheroma thickness, then an aneurysm is created.^[51]

Visible features

Severe atherosclerosis of the aorta. Autopsy specimen.

Although arteries are not typically studied microscopically, two plaque types can be distinguished:^[52]

1. The fibro-lipid (fibro-fatty) plaque is characterized by an accumulation of lipid-laden cells underneath the intima of the arteries, typically without narrowing the lumen due to compensatory expansion of the bounding muscular layer of the artery wall. Beneath the endothelium there is a "fibrous cap" covering the atheromatous "core" of the plaque. The core consists of lipid-laden cells (macrophages and smooth muscle cells) with elevated tissue cholesterol and cholesterol ester content, fibrin, proteoglycans, collagen, elastin, and cellular debris. In advanced plaques, the central core of the plaque usually contains extracellular cholesterol deposits (released from dead cells), which form areas of cholesterol crystals with empty, needle-like clefts. At the periphery of the plaque are younger "foamy" cells and capillaries. These plaques usually produce the most damage to the individual when they rupture. Cholesterol crystals may also play a role.^[53]
2. The fibrous plaque is also localized under the intima, within the wall of the artery resulting in thickening and expansion of the wall and, sometimes, spotty localized narrowing of the lumen with some atrophy of the muscular layer. The fibrous plaque contains collagen fibers (eosinophilic), precipitates of calcium (hematoxylinophilic) and, rarely, lipid-laden cells.

In effect, the muscular portion of the artery wall forms small aneurysms just large enough to hold the atheroma that are present. The muscular portion of artery walls usually remain strong, even after they have remodeled to compensate for the atheromatous plaques.

However, atheromas within the vessel wall are soft and fragile with little elasticity. Arteries constantly expand and contract with each heartbeat, i.e., the pulse. In addition, the calcification deposits between the outer portion of the atheroma and the muscular wall, as they progress, lead to a loss of elasticity and stiffening of the artery as a whole.

The calcification deposits,^[54] after they have become sufficiently advanced, are partially visible on coronary artery computed tomography or electron beam tomography (EBT) as rings of increased radiographic density, forming halos around the outer edges of the atheromatous plaques, within the artery wall. On CT, >130 units on the Hounsfield scale (some argue for 90 units) has been the radiographic density usually accepted as clearly representing tissue calcification within arteries. These deposits demonstrate unequivocal evidence of the disease, relatively advanced, even though the lumen of the artery is often still normal by angiography.

Rupture and stenosis

Progression of atherosclerosis to late complications.

Although the disease process tends to be slowly progressive over decades, it usually remains asymptomatic until an atheroma ulcerates, which leads to immediate blood clotting at the site of atheroma ulcer. This triggers a cascade of events that leads to clot enlargement, which may quickly obstruct the flow of blood. A complete blockage leads to ischemia of the myocardial (heart) muscle and damage. This process is the myocardial infarction or "heart attack".

If the heart attack is not fatal, fibrous organization of the clot within the lumen ensues, covering the rupture but also producing stenosis or closure of the lumen, or over time and after repeated ruptures, resulting in a persistent, usually localized stenosis or blockage of the artery lumen. Stenoses can be slowly progressive, whereas plaque ulceration is a sudden event that occurs specifically in atheromas with thinner/weaker fibrous caps that have become "unstable".

Repeated plaque ruptures, ones not resulting in total lumen closure, combined with the clot patch over the rupture and healing response to stabilize the clot is the process that produces most stenoses over time. The stenotic areas tend to become more stable despite increased flow velocities at these narrowings. Most major blood-flow-stopping events occur at large plaques, which, prior to their rupture, produced very little if any stenosis.

From clinical trials, 20% is the average stenosis at plaques that subsequently rupture with resulting complete artery closure. Most severe clinical events do not occur at plaques that produce high-grade stenosis. From clinical trials, only 14% of heart attacks occur from artery closure at plaques producing a 75% or greater stenosis prior to the vessel closing.^{[citation needed]}

If the fibrous cap separating a soft atheroma from the bloodstream within the artery ruptures, tissue fragments are exposed and released. These tissue fragments are very clot-promoting, containing collagen and tissue factor; they activate platelets and activate the system of coagulation. The result is the formation of a thrombus (blood clot) overlying the atheroma, which obstructs blood flow acutely. With the obstruction of blood flow, downstream tissues are starved of oxygen and nutrients. If this is the myocardium (heart muscle) angina (cardiac chest pain) or myocardial infarction (heart attack) develops.

Accelerated growth of plaques

The distribution of atherosclerotic plaques in a part of arterial endothelium is inhomogeneous. The multiple and focal development of atherosclerotic changes is similar to that in the development of amyloid plaques in the brain and that of age spots on the skin. Misrepair-accumulation aging theory suggests that misrepair mechanisms^[55][56] play an important role in the focal development of atherosclerosis.^[57] Development of a plaque is a result of repair of injured endothelium. Because of the infusion of lipids into sub-endothelium, the repair has to end up with altered remodeling of local endothelium. This is the manifestation of a misrepair. Important is this altered remodeling makes the local endothelium have increased fragility to damage and have reduced repair-efficiency. As a consequence, this part of endothelium has increased risk to be injured and to be misrepaired. Thus, the accumulation of misrepairs of endothelium is focalized and self-accelerating. In this way, the growing of a plaque is also self-accelerating. Within a part of arterial wall, the oldest plaque is always the biggest, and is the most dangerous one to cause blockage of local artery.

Components

The plaque is divided into three distinct components:

1. The atheroma ("lump of gruel", from Greek ἀθήρα (athera), meaning 'gruel'), which is the nodular accumulation of a soft, flaky, yellowish material at the center of large plaques, composed of macrophages nearest the lumen of the artery
2. Underlying areas of cholesterol crystals
3. Calcification at the outer base of older or more advanced lesions. Atherosclerotic lesions, or atherosclerotic plaques, are separated into two broad categories: Stable and unstable (also called vulnerable).^[58] The pathobiology of atherosclerotic lesions is very complicated, but generally, stable atherosclerotic plaques, which tend to be asymptomatic, are rich in extracellular matrix and smooth muscle cells. On the other hand, unstable plaques are rich in macrophages and foam cells, and the extracellular matrix separating the lesion from the arterial lumen (also known as the fibrous cap) is usually weak and prone to rupture.^[59] Ruptures of the fibrous cap expose thrombogenic material, such as collagen,^[60] to the circulation and eventually induce thrombus formation in the lumen. Upon formation, intraluminal thrombi can occlude arteries outright (e.g., coronary occlusion), but more often they detach, move into the circulation, and eventually occlude smaller downstream branches causing thromboembolism.

Apart from thromboembolism, chronically expanding atherosclerotic lesions can cause complete closure of the lumen. Chronically expanding lesions are often asymptomatic until lumen stenosis is so severe (usually over 80%) that blood supply to downstream tissue(s) is insufficient, resulting in ischemia. These complications of advanced atherosclerosis are chronic, slowly progressive and cumulative. Most commonly, soft plaque suddenly ruptures (see vulnerable plaque), causing the formation of a thrombus that will rapidly slow or stop blood flow, leading to death of the tissues fed by the artery in approximately five minutes. This event is called an infarction.

Diagnosis

Microphotography of arterial wall with calcified (violet color) atherosclerotic plaque (hematoxylin and eosin stain)

Areas of severe narrowing, stenosis, detectable by angiography, and to a lesser extent "stress testing" have long been the focus of human diagnostic techniques for cardiovascular disease, in general. However, these methods focus on detecting only severe narrowing, not the underlying atherosclerosis disease. As demonstrated by human clinical studies, most severe events occur in locations with heavy plaque, yet little or no lumen narrowing present before debilitating events suddenly occur. Plaque rupture can lead to artery lumen occlusion within seconds to minutes, and potential permanent debility and sometimes sudden death.

Plaques that have ruptured are called complicated plaques. The extracellular matrix of the lesion breaks, usually at the shoulder of the fibrous cap that separates the lesion from the arterial lumen, where the exposed thrombogenic components of the plaque, mainly collagen will trigger thrombus formation. The thrombus then travels downstream to other blood vessels, where the blood clot may partially or completely block blood flow. If the blood flow is completely blocked, cell deaths occur due to the lack of oxygen supply to nearby cells, resulting in necrosis. The narrowing or obstruction of blood flow can occur in any artery within the body. Obstruction of arteries supplying the heart muscle results in a heart attack, while the obstruction of arteries supplying the brain results in an ischaemic stroke.

Doppler ultrasound of right internal Carotid artery with calcified and non-calcified plaques showing less than 70% stenosis

Lumen stenosis that is greater than 75% was considered the hallmark of clinically significant disease in the past because recurring episodes of angina and abnormalities in stress tests are only detectable at that particular severity of stenosis. However, clinical trials have shown that only about 14% of clinically debilitating events occur at sites with more than 75% stenosis. The majority of cardiovascular events that involve sudden rupture of the atheroma plaque do not display any evident narrowing of the lumen. Thus, greater attention has been focused on "vulnerable plaque" from the late 1990s onwards.^[61]

Besides the traditional diagnostic methods such as angiography and stress-testing, other detection techniques have been developed in the past decades for earlier detection of atherosclerotic disease. Some of the detection approaches include anatomical detection and physiologic measurement.

Examples of anatomical detection methods include coronary calcium scoring by CT, carotid IMT (intimal media thickness) measurement by ultrasound, and intravascular ultrasound (IVUS). Examples of physiologic measurement methods include lipoprotein subclass analysis, HbA1c, hs-CRP, and homocysteine. Both anatomic and physiologic methods allow early detection before symptoms show up, disease staging and tracking of disease progression. Anatomic methods are more expensive and some of them are invasive in nature, such as IVUS. On the other hand, physiologic methods are often less expensive and safer. But they do not quantify the current state of the disease or directly track progression. In recent years, developments in nuclear imaging techniques such as PET and SPECT have provided ways of estimating the severity of atherosclerotic plaques.

Prevention

Up to 90% of cardiovascular disease may be preventable if established risk factors are avoided.^[62][63] Medical management of atherosclerosis first involves modification to risk factors–for example, via smoking cessation and diet restrictions. Additionally, a controlled exercise program combats atherosclerosis by improving circulation and functionality of the vessels. Exercise is also used to manage weight in patients who are obese, lower blood pressure, and decrease cholesterol. Often lifestyle modification is combined with medication therapy. For example, statins help to lower cholesterol, antiplatelet medications like aspirin help to prevent clots, and a variety of antihypertensive medications are routinely used to control blood pressure. If the combined efforts of risk factor modification and medication therapy are not sufficient to control symptoms, or fight imminent threats of ischemic events, a physician may resort to interventional or surgical procedures to correct the obstruction.^[64]

Combinations of statins, niacin and intestinal cholesterol absorption-inhibiting supplements (ezetimibe and others, and to a much lesser extent fibrates) have been the most successful in changing common but sub-optimal lipoprotein patterns and group outcomes. In the many secondary prevention and several primary prevention trials, several classes of lipoprotein-expression-altering (less correctly termed "cholesterol-lowering") agents have consistently reduced not only heart attack, stroke and hospitalization but also all-cause mortality rates. The first of the large secondary prevention comparative statin/placebo treatment trials was the Scandinavian Simvastatin Survival Study (4S)^[65] with over fifteen more studies extending through to the more recent ASTEROID^[66] trial published in 2006. The first primary prevention comparative treatment trial was AFCAPS/TexCAPS^[67] with multiple later comparative statin/placebo treatment trials including EXCEL,^[68] ASCOT^[69] and SPARCL.^[70][71] While the statin trials have all been clearly favorable for improved human outcomes, only ASTEROID and SATURN showed evidence of atherosclerotic regression (slight). Both human and animal trials that showed evidence of disease regression used more aggressive combination agent treatment strategies, which nearly always included niacin.^[72]

Treatment

Medical treatments often focus on alleviating symptoms. However measures which focus on decreasing underlying atherosclerosis—as opposed to simply treating symptoms—are more effective.^[73] Non-pharmaceutical means are usually the first method of treatment, such as stopping smoking and practicing regular exercise.^[74][75] If these methods do not work, medicines are usually the next step in treating cardiovascular diseases, and, with improvements, have increasingly become the most effective method over the long term.

The key to the more effective approaches is to combine multiple different treatment strategies.^[76] In addition, for those approaches, such as lipoprotein transport behaviors, which have been shown to produce the most success, adopting more aggressive combination treatment strategies taken on a daily basis and indefinitely has generally produced better results, both before and especially after people are symptomatic.^[73]

Diet

Changes in diet may help prevent the development of atherosclerosis. Tentative evidence suggests that a diet containing dairy products has no effect on or decreases the risk of cardiovascular disease.^[77][78]

A diet high in fruits and vegetables decreases the risk of cardiovascular disease and death.^[79] Evidence suggests that the Mediterranean diet may improve cardiovascular results.^[80] There is also evidence that a Mediterranean diet may be better than a low-fat diet in bringing about long-term changes to cardiovascular risk factors (e.g., lower cholesterol level and blood pressure).^[81]

Statins

The group of medications referred to as statins are widely prescribed for treating atherosclerosis. They have shown benefit in reducing cardiovascular disease and mortality in those with high cholesterol with few side effects.^[82]

These data are primarily in middle-age men and the conclusions are less clear for women and people over the age of 70.^[83]

Monocyte counts, as well as cholesterol markers such as LDL:HDL ratio and apolipiprotein B: apolipoprotein A-1 ratio can be used as markers to monitor the extent of atherosclerotic regression which proves useful in guiding patient treatments.^[84]

Surgery

When atherosclerosis has become severe and caused irreversible ischemia, such as tissue loss in the case of peripheral artery disease, surgery may be indicated. Vascular bypass surgery can re-establish flow around the diseased segment of artery, and angioplasty with or without stenting can reopen narrowed arteries and improve bloodflow. Coronary artery bypass grafting without manipulation of the ascending aorta has demonstrated reduced rates of postoperative stroke and mortality compared to traditional on-pump coronary revascularization.^[85]

Other

There is evidence that some anticoagulants, particularly warfarin, which inhibit clot formation by interfering with Vitamin K metabolism, may actually promote arterial calcification in the long term despite reducing clot formation in the short term.^[86][87][88]

Prognosis

Diabetics, despite not having clinically detectable atherosclerotic disease, have more severe debility from atherosclerotic events over time than non-diabetics who have already had atherosclerotic events. Thus diabetes has been upgraded to be viewed as an advanced atherosclerotic disease equivalent.

Research

Lipids

An indication of the role of HDL on atherosclerosis has been with the rare Apo-A1 Milano human genetic variant of this HDL protein. A small short-term trial using bacterial synthetized human Apo-A1 Milano HDL in people with unstable angina produced fairly dramatic reduction in measured coronary plaque volume in only six weeks vs. the usual increase in plaque volume in those randomized to placebo. The trial was published in JAMA in early 2006.^{[citation needed]} Ongoing work starting in the 1990s may lead to human clinical trials—probably by about 2008.^{[needs update]} These may use synthesized Apo-A1 Milano HDL directly, or they may use gene-transfer methods to pass the ability to synthesize the Apo-A1 Milano HDLipoprotein.^{[citation needed]}

Methods to increase high-density lipoprotein (HDL) particle concentrations, which in some animal studies largely reverses and remove atheromas, are being developed and researched.^{[citation needed]} However, increasing HDL by any means is not necessarily helpful. For example, the drug torcetrapib is the most effective agent currently known for raising HDL (by up to 60%). However, in clinical trials it also raised deaths by 60%. All studies regarding this drug were halted in December 2006.^[89]

The actions of macrophages drive atherosclerotic plaque progression. Immunomodulation of atherosclerosis is the term for techniques that modulate immune system function to suppress this macrophage action.^[90]

Research on genetic expression and control mechanisms is progressing. Topics include:

• PPAR, known to be important in blood sugar and variants of lipoprotein production and function;^{[citation needed]}
• The multiple variants of the proteins that form the lipoprotein transport particles.^{[citation needed]}

Involvement of lipid peroxidation chain reaction in atherogenesis^[91] triggered research on the protective role of the heavy isotope (deuterated) polyunsaturated fatty acids (D-PUFAs) that are less prone to oxidation than ordinary PUFAs (H-PUFAs). PUFAs are essential nutrients – they are involved in metabolism in that very form as they are consumed with food. In transgenic mice, that are a model for human-like lipoprotein metabolism, adding D-PUFAs to diet indeed reduced body weight gain, improved cholesterol handling and reduced atherosclerotic damage to aorta.^[92][93]

miRNA

MicroRNAs (miRNAs) have complementary sequences in the 3' UTR and 5' UTR of target mRNAs of protein-coding genes, and cause mRNA cleavage or repression of translational machinery. In diseased vascular vessels, miRNAs are dysregulated and highly expressed. miR-33 is found in cardiovascular diseases.^[94] It is involved in atherosclerotic initiation and progression including lipid metabolism, insulin signaling and glucose homeostatis, cell type progression and proliferation, and myeloid cell differentiation. It was found in rodents that the inhibition of miR-33 will raise HDL level and the expression of miR-33 is down-regulated in humans with atherosclerotic plaques.^{[95][96][97][98]}

miR-33a and miR-33b are located on intron 16 of human sterol regulatory element-binding protein 2 (SREBP2) gene on chromosome 22 and intron 17 of SREBP1 gene on chromosome 17.^[99] miR-33a/b regulates cholesterol/lipid homeostatis by binding in the 3’UTRs of genes involved in cholesterol transport such as ATP binding cassette (ABC) transporters and enhance or represses its expression. Study have shown that ABCA1 mediates transport of cholesterol from peripheral tissues to Apolipoprotein-1 and it is also important in the reverse cholesterol transport pathway, where cholesterol is delivered from peripheral tissue to the liver, where it can be excreted into bile or converted to bile acids prior to excretion.^[94] Therefore, we know that ABCA1 plays an important role in preventing cholesterol accumulation in macrophages. By enhancing miR-33 function, the level of ABCA1 is decreased, leading to decrease cellular cholesterol efflux to apoA-1. On the other hand, by inhibiting miR-33 function, the level of ABCA1 is increased and increases the cholesterol efflux to apoA-1. Suppression of miR-33 will lead to less cellular cholesterol and higher plasma HDL level through the regulation of ABCA1 expression.^[100]

The sugar, cyclodextrin, removed cholesterol that had built up in the arteries of mice fed a high-fat diet.^[101][102]

DNA damage

Aging is the most important risk factor for cardiovascular problems. The causative basis by which aging mediates its impact, independently of other recognized risk factors, remains to be determined. Evidence has been reviewed for a key role of DNA damage in vascular aging.^{[103][104][105]} 8-oxoG, a common type of oxidative damage in DNA, is found to accumulate in plaque vascular smooth muscle cells, macrophages and endothelial cells,^[106] thus linking DNA damage to plaque formation. DNA strand breaks also increased in atherosclerotic plaques.^[106] Werner syndrome (WS) is a premature aging condition in humans.^[107] WS is caused by a genetic defect in a RecQ helicase that is employed in several repair processes that remove damages from DNA. WS patients develop a considerable burden of atherosclerotic plaques in their coronary arteries and aorta: calcification of the aortic valve is also frequently observed.^[104] These findings link excessive unrepaired DNA damage to premature aging and early atherosclerotic plaque development (see DNA damage theory of aging).

Microorganisms

Microorganisms, living in the body (all together called microbiome), can contribute to atherosclerosis in many ways: modulation of the immune system, changes in metabolism, processing of nutrients and production of certain metabolites that can get into blood circulation.^[108] One of such metabolites, produced by gut bacteria, is trimethylamine N-oxide (TMAO). Its levels have been associated with atherosclerosis in human studies and animal research suggest that there can be a causal relation. An association between the bacterial genes encoding trimethylamine lyases — the enzymes involved in TMAO generation — and atherosclerosis has been noted.^[109][108]

Some controversial research has suggested a link between atherosclerosis and the presence of several different nanobacteria in the arteries, e.g., Chlamydophila pneumoniae,^{[citation needed]} though trials of current antibiotic treatments known to be usually effective in suppressing growth or killing these bacteria have not been successful in improving outcomes.

Apoptosis

From Wikipedia, the free encyclopedia

Apoptosis
An etoposide-treated DU145 prostate cancer cell exploding into a cascade of apoptotic bodies. The sub images were extracted from a 61-hour time-lapse microscopy video, created using quantitative phase-contrast microscopy. The optical thickness is color-coded. With increasing thickness, color changes from gray to yellow, red, purple and finally black.[1] See the video at The Cell: An Image Library

Apoptosis (from Ancient Greek ἀπόπτωσις "falling off") is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decay. The average adult human loses between 50 and 70 billion cells each day due to apoptosis. For an average human child between the ages of 8 and 14, approximately 20 to 30 billion cells die a day.

In contrast to necrosis, which is a form of traumatic cell death that results from acute cellular injury, apoptosis is a highly regulated and controlled process that confers advantages during an organism's lifecycle. For example, the separation of fingers and toes in a developing human embryo occurs because cells between the digits undergo apoptosis. Unlike necrosis, apoptosis produces cell fragments called apoptotic bodies that phagocytic cells are able to engulf and remove before the contents of the cell can spill out onto surrounding cells and cause damage to them.^[5]

Because apoptosis cannot stop once it has begun, it is a highly regulated process. Apoptosis can be initiated through one of two pathways. In the intrinsic pathway the cell kills itself because it senses cell stress, while in the extrinsic pathway the cell kills itself because of signals from other cells. Both pathways induce cell death by activating caspases, which are proteases, or enzymes that degrade proteins. The two pathways both activate initiator caspases, which then activate executioner caspases, which then kill the cell by degrading proteins indiscriminately.

Research on apoptosis has increased substantially since the early 1990s. In addition to its importance as a biological phenomenon, defective apoptotic processes have been implicated in a wide variety of diseases. Excessive apoptosis causes atrophy, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer. Some factors like Fas receptors and caspases promote apoptosis, while some members of the Bcl-2 family of proteins inhibit apoptosis.

Discovery and etymology

German scientist Karl Vogt was first to describe the principle of apoptosis in 1842. In 1885, anatomist Walther Flemming delivered a more precise description of the process of programmed cell death. However, it was not until 1965 that the topic was resurrected. While studying tissues using electron microscopy, John Foxton Ross Kerr at the University of Queensland was able to distinguish apoptosis from traumatic cell death.^[6] Following the publication of a paper describing the phenomenon, Kerr was invited to join Alastair R. Currie, as well as Andrew Wyllie, who was Currie's graduate student,^[7] at University of Aberdeen. In 1972, the trio published a seminal article in the British Journal of Cancer.^[8] Kerr had initially used the term programmed cell necrosis, but in the article, the process of natural cell death was called apoptosis. Kerr, Wyllie and Currie credited James Cormack, a professor of Greek language at University of Aberdeen, with suggesting the term apoptosis. Kerr received the Paul Ehrlich and Ludwig Darmstaedter Prize on March 14, 2000, for his description of apoptosis. He shared the prize with Boston biologist H. Robert Horvitz.^[9]

For many years, neither "apoptosis" nor "programmed cell death" was a highly cited term. Two discoveries brought cell death from obscurity to a major field of research: identification of components of the cell death control and effector mechanisms, and linkage of abnormalities in cell death to human disease, in particular cancer.

The 2002 Nobel Prize in Medicine was awarded to Sydney Brenner, Horvitz and John E. Sulston for their work identifying genes that control apoptosis. The genes were identified by studies in the nematode C. elegans and homologues of these genes function in humans to regulate apoptosis.

John E. Sulston won the Nobel Prize in Medicine in 2002, for his pioneering research on apoptosis.

In Greek, apoptosis translates to the "falling off" of leaves from a tree.^[10] Cormack, professor of Greek language, reintroduced the term for medical use as it had a medical meaning for the Greeks over two thousand years before. Hippocrates used the term to mean "the falling off of the bones".  Galen extended its meaning to "the dropping of the scabs". Cormack was no doubt aware of this usage when he suggested the name. Debate continues over the correct pronunciation, with opinion divided between a pronunciation with the second p silent (/æpəˈtoʊsɪs/ ap-ə-TOH-sis^[11][12]) and the second p pronounced (/eɪpəpˈtoʊsɪs/),^[11][13] as in the original Greek.^{[citation needed]} In English, the p of the Greek -pt- consonant cluster is typically silent at the beginning of a word (e.g. pterodactyl, Ptolemy), but articulated when used in combining forms preceded by a vowel, as in helicopter or the orders of insects: diptera, lepidoptera, etc.

In the original Kerr, Wyllie & Currie paper,^[8] there is a footnote regarding the pronunciation:

"We are most grateful to Professor James Cormack of the Department of Greek, University of Aberdeen, for suggesting this term. The word "apoptosis" (ἀπόπτωσις) is used in Greek to describe the "dropping off" or "falling off" of petals from flowers, or leaves from trees. To show the derivation clearly, we propose that the stress should be on the penultimate syllable, the second half of the word being pronounced like "ptosis" (with the "p" silent), which comes from the same root "to fall", and is already used to describe the drooping of the upper eyelid."

Activation mechanisms

Control Of The Apoptotic Mechanisms

The initiation of apoptosis is tightly regulated by activation mechanisms, because once apoptosis has begun, it inevitably leads to the death of the cell.^{[14] [15]} The two best-understood activation mechanisms are the intrinsic pathway (also called the mitochondrial pathway) and the extrinsic pathway.^[16] The intrinsic pathway is activated by intracellular signals generated when cells are stressed and depends on the release of proteins from the intermembrane space of mitochondria.^[17] The extrinsic pathway is activated by extracellular ligands binding to cell-surface death receptors, which leads to the formation of the death-inducing signaling complex (DISC).^[18]

A cell initiates intracellular apoptotic signaling in response to a stress, which may bring about cell suicide. The binding of nuclear receptors by glucocorticoids,^[19] heat,^[19] radiation,^[19] nutrient deprivation,^[19] viral infection,^[19] hypoxia^[19] and increased intracellular calcium concentration,^[20][21] for example, by damage to the membrane, can all trigger the release of intracellular apoptotic signals by a damaged cell. A number of cellular components, such as poly ADP ribose polymerase, may also help regulate apoptosis.^[22]

Before the actual process of cell death is precipitated by enzymes, apoptotic signals must cause regulatory proteins to initiate the apoptosis pathway. This step allows those signals to cause cell death, or the process to be stopped, should the cell no longer need to die. Several proteins are involved, but two main methods of regulation have been identified: the targeting of mitochondria functionality,^[23] or directly transducing the signal via adaptor proteins to the apoptotic mechanisms. An extrinsic pathway for initiation identified in several toxin studies is an increase in calcium concentration within a cell caused by drug activity, which also can cause apoptosis via a calcium binding protease calpain.

Intrinsic pathway

The mitochondria are essential to multicellular life. Without them, a cell ceases to respire aerobically and quickly dies. This fact forms the basis for some apoptotic pathways. Apoptotic proteins that target mitochondria affect them in different ways. They may cause mitochondrial swelling through the formation of membrane pores, or they may increase the permeability of the mitochondrial membrane and cause apoptotic effectors to leak out.^[19][24] They are very closely related to intrinsic pathway, and tumors arise more frequently through intrinsic pathway than the extrinsic pathway because of sensitivity.^[25] There is also a growing body of evidence indicating that nitric oxide is able to induce apoptosis by helping to dissipate the membrane potential of mitochondria and therefore make it more permeable.^[26] Nitric oxide has been implicated in initiating and inhibiting apoptosis through its possible action as a signal molecule of subsequent pathways that activate apoptosis.

Mitochondrial proteins known as SMACs (second mitochondria-derived activator of caspases) are released into the cell's cytosol following the increase in permeability of the mitochondria membranes. SMAC binds to proteins that inhibit apoptosis (IAPs) thereby deactivating them, and preventing the IAPs from arresting the process and therefore allowing apoptosis to proceed. IAP also normally suppresses the activity of a group of cysteine proteases called caspases,^[28] which carry out the degradation of the cell. Therefore, the actual degradation enzymes can be seen to be indirectly regulated by mitochondrial permeability.

Cytochrome c is also released from mitochondria due to formation of a channel, the mitochondrial apoptosis-induced channel (MAC), in the outer mitochondrial membrane,^[29] and serves a regulatory function as it precedes morphological change associated with apoptosis.^[19] Once cytochrome c is released it binds with Apoptotic protease activating factor – 1 (Apaf-1) and ATP, which then bind to pro-caspase-9 to create a protein complex known as an apoptosome. The apoptosome cleaves the pro-caspase to its active form of caspase-9, which in turn activates the effector caspase-3.

MAC (not to be confused with the membrane attack complex formed by complement activation, also commonly denoted as MAC), also called "Mitochondrial Outer Membrane Permeabilization Pore" is regulated by various proteins, such as those encoded by the mammalian Bcl-2 family of anti-apoptopic genes, the homologs of the ced-9 gene found in C. elegans.^[30][31] Bcl-2 proteins are able to promote or inhibit apoptosis by direct action on MAC/MOMPP. Bax and/or Bak form the pore, while Bcl-2, Bcl-xL or Mcl-1 inhibit its formation.

Extrinsic pathway

Overview of signal transduction pathways.

Overview of TNF (middle) and Fas (bottom)

signalling in apoptosis, an example of direct

signal transduction.

Two theories of the direct initiation of apoptotic mechanisms in mammals have been suggested: the TNF-induced (tumor necrosis factor) model and the Fas-Fas ligand-mediated model, both involving receptors of the TNF receptor (TNFR) family^[32] coupled to extrinsic signals.
TNF path

TNF-alpha is a cytokine produced mainly by activated macrophages, and is the major extrinsic mediator of apoptosis. Most cells in the human body have two receptors for TNF-alpha: TNFR1 and TNFR2. The binding of TNF-alpha to TNFR1 has been shown to initiate the pathway that leads to caspase activation via the intermediate membrane proteins TNF receptor-associated death domain (TRADD) and Fas-associated death domain protein (FADD). cIAP1/2 can inhibit TNF-α signaling by binding to TRAF2. FLIP inhibits the activation of caspase-8.^[33] Binding of this receptor can also indirectly lead to the activation of transcription factors involved in cell survival and inflammatory responses.^[34] However, signalling through TNFR1 might also induce apoptosis in a caspase-independent manner.^[35] The link between TNF-alpha and apoptosis shows why an abnormal production of TNF-alpha plays a fundamental role in several human diseases, especially in autoimmune diseases.

Fas path

The fas receptor (First apoptosis signal) – (also known as Apo-1 or CD95) is a transmembrane protein of the TNF family which binds the Fas ligand (FasL).^[32] The interaction between Fas and FasL results in the formation of the death-inducing signaling complex (DISC), which contains the FADD, caspase-8 and caspase-10. In some types of cells (type I), processed caspase-8 directly activates other members of the caspase family, and triggers the execution of apoptosis of the cell. In other types of cells (type II), the Fas-DISC starts a feedback loop that spirals into increasing release of proapoptotic factors from mitochondria and the amplified activation of caspase-8.^[36]

Common components

Following TNF-R1 and Fas activation in mammalian cells a balance between proapoptotic (BAX,^[37] BID, BAK, or BAD) and anti-apoptotic (Bcl-Xl and Bcl-2) members of the Bcl-2 family are established. This balance is the proportion of proapoptotic homodimers that form in the outer-membrane of the mitochondrion. The proapoptotic homodimers are required to make the mitochondrial membrane permeable for the release of caspase activators such as cytochrome c and SMAC. Control of proapoptotic proteins under normal cell conditions of nonapoptotic cells is incompletely understood, but in general, Bax or Bak are activated by the activation of BH3-only proteins, part of the Bcl-2 family.

Caspases Caspases play the central role in the transduction of ER apoptotic signals. Caspases are proteins that are highly conserved, cysteine-dependent aspartate-specific proteases. There are two types of caspases: initiator caspases, caspase 2,8,9,10,11,12, and effector caspases, caspase 3,6,7. The activation of initiator caspases requires binding to specific oligomeric activator protein. Effector caspases are then activated by these active initiator caspases through proteolytic cleavage. The active effector caspases then proteolytically degrade a host of intracellular proteins to carry out the cell death program.

Caspase-independent apoptotic pathway There also exists a caspase-independent apoptotic pathway that is mediated by AIF (apoptosis-inducing factor).^[38]

Apoptosis model in amphibians

Amphibian Metamorphosis

Amphibian frog Xenopus laevis serves as an ideal model system for the study of the mechanisms of apoptosis. In fact, iodine and thyroxine also stimulate the spectacular apoptosis of the cells of the larval gills, tail and fins in amphibians metamorphosis, and stimulate the evolution of their nervous system transforming the aquatic, vegetarian tadpole into the terrestrial, carnivorous frog.

Negative regulators of apoptosis

Negative regulation of apoptosis inhibits cell death signaling pathways, helping tumors to evade cell death and developing drug resistance. Many families of proteins act as negative regulators categorized into either antiapoptotic factors, such as IAPs and Bcl-2 proteins or prosurvival factors like cFLIP, BNIP3, FADD, Akt, and NF-κB ^[43].

Proteolytic caspase cascade: Killing the cell

Many pathways and signals lead to apoptosis, but these converge on a single mechanism that actually causes the death of the cell. After a cell receives stimulus, it undergoes organized degradation of cellular organelles by activated proteolytic caspases. In addition to the destruction of cellular organelles, mRNA is rapidly and globally degraded by a mechanism that is not yet fully characterized.^[44] mRNA decay is triggered very early in apoptosis.

A cell undergoing apoptosis shows a series of characteristic morphological changes. Early alterations include:

1. Cell shrinkage and rounding occur because of the retraction lamellipodia and the breakdown of the proteinaceous cytoskeleton by caspases.^[45]
2. The cytoplasm appears dense, and the organelles appear tightly packed.
3. Chromatin undergoes condensation into compact patches against the nuclear envelope (also known as the perinuclear envelope) in a process known as pyknosis, a hallmark of apoptosis.^[46][47]
4. The nuclear envelope becomes discontinuous and the DNA inside it is fragmented in a process referred to as karyorrhexis. The nucleus breaks into several discrete chromatin bodies or nucleosomal units due to the degradation of DNA.^[48]

Apoptosis progresses quickly and its products are quickly removed, making it difficult to detect or visualize on classical histology sections. During karyorrhexis, endonuclease activation leaves short DNA fragments, regularly spaced in size. These give a characteristic "laddered" appearance on agar gel after electrophoresis.^[49] Tests for DNA laddering differentiate apoptosis from ischemic or toxic cell death.^[50]

Apoptotic cell disassembly

Different steps in apoptotic cell disassembly.^[51]

Before the apoptotic cell is disposed of, there is a
process of disassembly. There are three recognized
steps in apoptotic cell disassembly:^[52]

1. Membrane blebbing: The cell membrane shows irregular buds known as blebs. Initially these are smaller surface blebs. Later these can grow into larger so-called dynamic membrane blebs.^[52] An important regulator of apoptotic cell membrane blebbing is ROCK1 (rho associated coiled-coil-containing protein kinase 1).^[53][54]
2. Formation of membrane protrusions: Some cell types, under specific conditions, may develop different types of long, thin extensions of the cell membrane called membrane protrusions. Three types have been described: microtubule spikes, apoptopodia (feet of death), and beaded apoptopodia (the latter having a beads-on-a-string appearance).^[55][56][57] Pannexin 1 is an important component of membrane channels involved in the formation of apoptopodia and beaded apoptopodia.^[56]
3. Fragmentation: The cell breaks apart into multiple vesicles called apoptotic bodies, which undergo phagocytosis. The plasma membrane protrusions may help bring apoptotic bodies closer to phagocytes.

Removal of dead cells

The removal of dead cells by neighboring phagocytic cells has been termed efferocytosis.^[58] Dying cells that undergo the final stages of apoptosis display phagocytotic molecules, such as phosphatidylserine, on their cell surface.^[59] Phosphatidylserine is normally found on the inner leaflet surface of the plasma membrane, but is redistributed during apoptosis to the extracellular surface by a protein known as scramblase.^[60] These molecules mark the cell for phagocytosis by cells possessing the appropriate receptors, such as macrophages.^[61] The removal of dying cells by phagocytes occurs in an orderly manner without eliciting an inflammatory response.^[62] During apoptosis cellular RNA and DNA are separated from each other and sorted to different apoptotic bodies; separation of RNA is initiated as nucleolar segregation.^[63]

Pathway knock-outs

Many knock-outs have been made in the apoptosis pathways to test the function of each of the proteins. Several caspases, in addition to APAF1 and FADD, have been mutated to determine the new phenotype. In order to create a tumor necrosis factor (TNF) knockout, an exon containing the nucleotides 3704–5364 was removed from the gene. This exon encodes a portion of the mature TNF domain, as well as the leader sequence, which is a highly conserved region necessary for proper intracellular processing. TNF-/- mice develop normally and have no gross structural or morphological abnormalities. However, upon immunization with SRBC (sheep red blood cells), these mice demonstrated a deficiency in the maturation of an antibody response; they were able to generate normal levels of IgM, but could not develop specific IgG levels. Apaf-1 is the protein that turns on caspase 9 by cleavage to begin the caspase cascade that leads to apoptosis. Since a -/- mutation in the APAF-1 gene is embryonic lethal, a gene trap strategy was used in order to generate an APAF-1 -/- mouse. This assay is used to disrupt gene function by creating an intragenic gene fusion. When an APAF-1 gene trap is introduced into cells, many morphological changes occur, such as spina bifida, the persistence of interdigital webs, and open brain. In addition, after embryonic day 12.5, the brain of the embryos showed several structural changes. APAF-1 cells are protected from apoptosis stimuli such as irradiation. A BAX-1 knock-out mouse exhibits normal forebrain formation and a decreased programmed cell death in some neuronal populations and in the spinal cord, leading to an increase in motor neurons.

The caspase proteins are integral parts of the apoptosis pathway, so it follows that knock-outs made have varying damaging results. A caspase 9 knock-out leads to a severe brain malformation. A caspase 8 knock-out leads to cardiac failure and thus embryonic lethality. However, with the use of cre-lox technology, a caspase 8 knock-out has been created that exhibits an increase in peripheral T cells, an impaired T cell response, and a defect in neural tube closure. These mice were found to be resistant to apoptosis mediated by CD95, TNFR, etc. but not resistant to apoptosis caused by UV irradiation, chemotherapeutic drugs, and other stimuli. Finally, a caspase 3 knock-out was characterized by ectopic cell masses in the brain and abnormal apoptotic features such as membrane blebbing or nuclear fragmentation. A remarkable feature of these KO mice is that they have a very restricted phenotype: Casp3, 9, APAF-1 KO mice have deformations of neural tissue and FADD and Casp 8 KO showed defective heart development, however in both types of KO other organs developed normally and some cell types were still sensitive to apoptotic stimuli suggesting that unknown proapoptotic pathways exist.

Methods for distinguishing apoptotic from necrotic (necroptotic) cells

In order to perform analysis of apoptotic versus necrotic (necroptotic) cells, one can do analysis of morphology by time-lapse microscopy, flow fluorocytometry, and transmission electron microscopy. There are also various biochemical techniques for analysis of cell surface markers (phosphatidylserine exposure versus cell permeability by flow cytometry), cellular markers such as DNA fragmentation^[64] (flow cytometry),^[65] caspase activation, Bid cleavage, and cytochrome c release (Western blotting). It is important to know how primary and secondary necrotic cells can be distinguished by analysis of supernatant for caspases, HMGB1, and release of cytokeratin 18. However, no distinct surface or biochemical markers of necrotic cell death have been identified yet, and only negative markers are available. These include absence of apoptotic markers (caspase activation, cytochrome c release, and oligonucleosomal DNA fragmentation) and differential kinetics of cell death markers (phosphatidylserine exposure and cell membrane permeabilization). A selection of techniques that can be used to distinguish apoptosis from necroptotic cells could be found in these references.

Implication in disease

A section of mouse liver showing several apoptotic cells, indicated by arrows

A section of mouse liver stained to show cells undergoing apoptosis (orange)

Neonatal cardiomyocytes ultrastructure after anoxia-reoxygenation.

Defective pathways

The many different types of apoptotic pathways contain a multitude of different biochemical components, many of them not yet understood.^[70] As a pathway is more or less sequential in nature, removing or modifying one component leads to an effect in another. In a living organism, this can have disastrous effects, often in the form of disease or disorder. A discussion of every disease caused by modification of the various apoptotic pathways would be impractical, but the concept overlying each one is the same: The normal functioning of the pathway has been disrupted in such a way as to impair the ability of the cell to undergo normal apoptosis. This results in a cell that lives past its "use-by-date" and is able to replicate and pass on any faulty machinery to its progeny, increasing the likelihood of the cell's becoming cancerous or diseased.

A recently described example of this concept in action can be seen in the development of a lung cancer called NCI-H460.^[71] The X-linked inhibitor of apoptosis protein (XIAP) is overexpressed in cells of the H460 cell line. XIAPs bind to the processed form of caspase-9, and suppress the activity of apoptotic activator cytochrome c, therefore overexpression leads to a decrease in the amount of proapoptotic agonists. As a consequence, the balance of anti-apoptotic and proapoptotic effectors is upset in favour of the former, and the damaged cells continue to replicate despite being directed to die. Defects in regulation of apoptosis in cancer cells occur often at the level of control of transcription factors. As a particular example, defects in molecules that control transcription factor NF-κB in cancer change the mode of transcriptional regulation and the response to apoptotic signals, to curtail dependence on the tissue that the cell belongs. This degree of independence from external survival signals, can enable cancer metastasis.^[72]

Dysregulation of p53

The tumor-suppressor protein p53 accumulates when DNA is damaged due to a chain of biochemical factors. Part of this pathway includes alpha-interferon and beta-interferon, which induce transcription of the p53 gene, resulting in the increase of p53 protein level and enhancement of cancer cell-apoptosis.^[73] p53 prevents the cell from replicating by stopping the cell cycle at G1, or interphase, to give the cell time to repair, however it will induce apoptosis if damage is extensive and repair efforts fail.^[74] Any disruption to the regulation of the p53 or interferon genes will result in impaired apoptosis and the possible formation of tumors.

Inhibition

Inhibition of apoptosis can result in a number of cancers, autoimmune diseases, inflammatory diseases, and viral infections. It was originally believed that the associated accumulation of cells was due to an increase in cellular proliferation, but it is now known that it is also due to a decrease in cell death. The most common of these diseases is cancer, the disease of excessive cellular proliferation, which is often characterized by an overexpression of IAP family members. As a result, the malignant cells experience an abnormal response to apoptosis induction: Cycle-regulating genes (such as p53, ras or c-myc) are mutated or inactivated in diseased cells, and further genes (such as bcl-2) also modify their expression in tumors. Some apoptotic factors are vital during mitochondrial respiration e.g. cytochrome C.^[75] Pathological inactivation of apoptosis in cancer cells is correlated with frequent respiratory metabolic shifts toward glycolysis (an observation known as the “Warburg hypothesis” ^[76]).

HeLa cell

Apoptosis in HeLa^[b] cells is inhibited by proteins produced by the cell; these inhibitory proteins target retinoblastoma tumor-suppressing proteins.^[77] These tumor-suppressing proteins regulate the cell cycle, but are rendered inactive when bound to an inhibitory protein.^[77] HPV E6 and E7 are inhibitory proteins expressed by the human papillomavirus, HPV being responsible for the formation of the cervical tumor from which HeLa cells are derived.^[78] HPV E6 causes p53, which regulates the cell cycle, to become inactive.^[79] HPV E7 binds to retinoblastoma tumor suppressing proteins and limits its ability to control cell division.^[79] These two inhibitory proteins are partially responsible for HeLa cells' immortality by inhibiting apoptosis to occur.^[80] CDV (Canine Distemper Virus) is able to induce apoptosis despite the presence of these inhibitory proteins. This is an important oncolytic property of CDV: this virus is capable of killing canine lymphoma cells. Oncoproteins E6 and E7 still leave p53 inactive, but they are not able to avoid the activation of caspases induced from the stress of viral infection. These oncolytic properties provided a promising link between CDV and lymphoma apoptosis, which can lead to development of alternative treatment methods for both canine lymphoma and human non-Hodgkin lymphoma. Defects in the cell cycle are thought to be responsible for the resistance to chemotherapy or radiation by certain tumor cells, so a virus that can induce apoptosis despite defects in the cell cycle is useful for cancer treatment.^[80]

Treatments

The main method of treatment for death from signaling-related diseases involves either increasing or decreasing the susceptibility of apoptosis in diseased cells, depending on whether the disease is caused by either the inhibition of or excess apoptosis. For instance, treatments aim to restore apoptosis to treat diseases with deficient cell death, and to increase the apoptotic threshold to treat diseases involved with excessive cell death. To stimulate apoptosis, one can increase the number of death receptor ligands (such as TNF or TRAIL), antagonize the anti-apoptotic Bcl-2 pathway, or introduce Smac mimetics to inhibit the inhibitor (IAPs). The addition of agents such as Herceptin, Iressa, or Gleevec works to stop cells from cycling and causes apoptosis activation by blocking growth and survival signaling further upstream. Finally, adding p53-MDM2 complexes displaces p53 and activates the p53 pathway, leading to cell cycle arrest and apoptosis. Many different methods can be used either to stimulate or to inhibit apoptosis in various places along the death signaling pathway.^[81]

Apoptosis is a multi-step, multi-pathway cell-death programme that is inherent in every cell of the body. In cancer, the apoptosis cell-division ratio is altered. Cancer treatment by chemotherapy and irradiation kills target cells primarily by inducing apoptosis.

Hyperactive apoptosis

On the other hand, loss of control of cell death (resulting in excess apoptosis) can lead to neurodegenerative diseases, hematologic diseases, and tissue damage. It is to note that neurons that rely on mitochondrial respiration undergo apoptosis in neurodegenerative diseases such as Alzheimer’s ^[82] and Parkinson’s ^[83] (an observation known as the “Inverse Warburg hypothesis”). Moreover, there is an inverse epidemiological comorbidity between neurodegenerative diseases and cancer.^[85] The progression of HIV is directly linked to excess, unregulated apoptosis. In a healthy individual, the number of CD4+ lymphocytes is in balance with the cells generated by the bone marrow; however, in HIV-positive patients, this balance is lost due to an inability of the bone marrow to regenerate CD4+ cells. In the case of HIV, CD4+ lymphocytes die at an accelerated rate through uncontrolled apoptosis, when stimulated. At the molecular level, hyperactive apoptosis can be caused by defects in signaling pathways that regulate the Bcl-2 family proteins. Increased expression of apoptotic proteins such as BIM, or their decreased proteolysis, leads to cell death, and can cause a number of pathologies, depending on the cells where excessive activity of BIM occurs. Cancer cells can escape apoptosis through mechanisms that suppress BIM expression or by increased proteolysis of BIM.^[86]

Treatments

Treatments aiming to inhibit works to block specific caspases. Finally, the Akt protein kinase promotes cell survival through two pathways. Akt phosphorylates and inhibits Bad (a Bcl-2 family member), causing Bad to interact with the 14-3-3 scaffold, resulting in Bcl dissociation and thus cell survival. Akt also activates IKKα, which leads to NF-κB activation and cell survival. Active NF-κB induces the expression of anti-apoptotic genes such as Bcl-2, resulting in inhibition of apoptosis. NF-κB has been found to play both an antiapoptotic role and a proapoptotic role depending on the stimuli utilized and the cell type.^[87]

HIV progression

The progression of the human immunodeficiency virus infection into AIDS is due primarily to the depletion of CD4+ T-helper lymphocytes in a manner that is too rapid for the body's bone marrow to replenish the cells, leading to a compromised immune system. One of the mechanisms by which T-helper cells are depleted is apoptosis, which results from a series of biochemical pathways:^[88]

1. HIV enzymes deactivate anti-apoptotic Bcl-2. This does not directly cause cell death but primes the cell for apoptosis should the appropriate signal be received. In parallel, these enzymes activate proapoptotic procaspase-8, which does directly activate the mitochondrial events of apoptosis.
2. HIV may increase the level of cellular proteins that prompt Fas-mediated apoptosis.
3. HIV proteins decrease the amount of CD4 glycoprotein marker present on the cell membrane.
4. Released viral particles and proteins present in extracellular fluid are able to induce apoptosis in nearby "bystander" T helper cells.
5. HIV decreases the production of molecules involved in marking the cell for apoptosis, giving the virus time to replicate and continue releasing apoptotic agents and virions into the surrounding tissue.
6. The infected CD4+ cell may also receive the death signal from a cytotoxic T cell.

Cells may also die as direct consequences of viral infections. HIV-1 expression induces tubular cell G2/M arrest and apoptosis.^[89] The progression from HIV to AIDS is not immediate or even necessarily rapid; HIV's cytotoxic activity toward CD4+ lymphocytes is classified as AIDS once a given patient's CD4+ cell count falls below 200.^[90]

Researchers from Kumamoto University in Japan have developed a new method to eradicate HIV in viral reservoir cells, named "Lock-in and apoptosis." Using the synthesized compound Heptanoylphosphatidyl L-Inositol Pentakisphophate (or L-Hippo) to bind strongly to the HIV protein PR55Gag, they were able to suppress viral budding. By suppressing viral budding, the researchers were able to trap the HIV virus in the cell and allow for the cell to undergo apoptosis (natural cell death). Associate Professor Mikako Fujita has stated that the approach is not yet available to HIV patients because the research team has to conduct further research on combining the drug therapy that currently exists with this "Lock-in and apoptosis" approach to lead to complete recovery from HIV.^[91]

Viral infection

Viral induction of apoptosis occurs when one or several cells of a living organism are infected with a virus, leading to cell death. Cell death in organisms is necessary for the normal development of cells and the cell cycle maturation.^[92] It is also important in maintaining the regular functions and activities of cells.

Viruses can trigger apoptosis of infected cells via a range of mechanisms including:

• Receptor binding
• Activation of protein kinase R (PKR)
• Interaction with p53
• Expression of viral proteins coupled to MHC proteins on the surface of the infected cell, allowing recognition by cells of the immune system (such as Natural Killer and cytotoxic T cells) that then induce the infected cell to undergo apoptosis.^[93]

Canine distemper virus (CDV) is known to cause apoptosis in central nervous system and lymphoid tissue of infected dogs in vivo and in vitro.^[94] Apoptosis caused by CDV is typically induced via the extrinsic pathway, which activates caspases that disrupt cellular function and eventually leads to the cells death.^[77] In normal cells, CDV activates caspase-8 first, which works as the initiator protein followed by the executioner protein caspase-3.^[77] However, apoptosis induced by CDV in HeLa cells does not involve the initiator protein caspase-8. HeLa cell apoptosis caused by CDV follows a different mechanism than that in vero cell lines.^[77] This change in the caspase cascade suggests CDV induces apoptosis via the intrinsic pathway, excluding the need for the initiator caspase-8. The executioner protein is instead activated by the internal stimuli caused by viral infection not a caspase cascade.^[77]

The Oropouche virus (OROV) is found in the family Bunyaviridae. The study of apoptosis brought on by Bunyaviridae was initiated in 1996, when it was observed that apoptosis was induced by the La Crosse virus into the kidney cells of baby hamsters and into the brains of baby mice.^[95]

OROV is a disease that is transmitted between humans by the biting midge (Culicoides paraensis).^[96] It is referred to as a zoonotic arbovirus and causes febrile illness, characterized by the onset of a sudden fever known as Oropouche fever.^[97]

The Oropouche virus also causes disruption in cultured cells – cells that are cultivated in distinct and specific conditions. An example of this can be seen in HeLa cells, whereby the cells begin to degenerate shortly after they are infected.^[95]

With the use of gel electrophoresis, it can be observed that OROV causes DNA fragmentation in HeLa cells. It can be interpreted by counting, measuring, and analyzing the cells of the Sub/G1 cell population.^[95] When HeLA cells are infected with OROV, the cytochrome C is released from the membrane of the mitochondria, into the cytosol of the cells. This type of interaction shows that apoptosis is activated via an intrinsic pathway.^[92]

In order for apoptosis to occur within OROV, viral uncoating, viral internalization, along with the replication of cells is necessary. Apoptosis in some viruses is activated by extracellular stimuli. However, studies have demonstrated that the OROV infection causes apoptosis to be activated through intracellular stimuli and involves the mitochondria.^[95]

Many viruses encode proteins that can inhibit apoptosis.^[98] Several viruses encode viral homologs of Bcl-2. These homologs can inhibit proapoptotic proteins such as BAX and BAK, which are essential for the activation of apoptosis. Examples of viral Bcl-2 proteins include the Epstein-Barr virus BHRF1 protein and the adenovirus E1B 19K protein.^[99] Some viruses express caspase inhibitors that inhibit caspase activity and an example is the CrmA protein of cowpox viruses. Whilst a number of viruses can block the effects of TNF and Fas. For example, the M-T2 protein of myxoma viruses can bind TNF preventing it from binding the TNF receptor and inducing a response.^[100] Furthermore, many viruses express p53 inhibitors that can bind p53 and inhibit its transcriptional transactivation activity. As a consequence, p53 cannot induce apoptosis, since it cannot induce the expression of proapoptotic proteins. The adenovirus E1B-55K protein and the hepatitis B virus HBx protein are examples of viral proteins that can perform such a function.^[101]

Viruses can remain intact from apoptosis in particular in the latter stages of infection. They can be exported in the apoptotic bodies that pinch off from the surface of the dying cell, and the fact that they are engulfed by phagocytes prevents the initiation of a host response. This favours the spread of the virus.^[100]

Plants

Programmed cell death in plants has a number of molecular similarities to that of animal apoptosis, but it also has differences, notable ones being the presence of a cell wall and the lack of an immune system that removes the pieces of the dead cell. Instead of an immune response, the dying cell synthesizes substances to break itself down and places them in a vacuole that ruptures as the cell dies. Whether this whole process resembles animal apoptosis closely enough to warrant using the name apoptosis (as opposed to the more general programmed cell death) is unclear.^[102]

Caspase-independent apoptosis

The characterization of the caspases allowed the development of caspase inhibitors, which can be used to determine whether a cellular process involves active caspases. Using these inhibitors it was discovered that cells can die while displaying a morphology similar to apoptosis without caspase activation.^[103] Later studies linked this phenomenon to the release of AIF (apoptosis-inducing factor) from the mitochondria and its translocation into the nucleus mediated by its NLS (nuclear localization signal). Inside the mitochondria, AIF is anchored to the inner membrane. In order to be released, the protein is cleaved by a calcium-dependent calpain protease.

Apoptosis protein subcellular location prediction

In 2003, a method was developed for predicting subcellular location of apoptosis proteins.^[104] Subsequent to this, various modes of Chou's pseudo amino acid composition were developed for improving the quality of predicting subcellular localization of apoptosis proteins based on their sequence information alone.