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Review of literature CORONARY ARTERY DISEASE Ischemic heart disease (which includes myocardial infarction, angina pectoris and heart failure when preceded by myocardial infarction) was the leading cause of death for both men and women worldwide. Important risk factors are previous cardiovascular disease, older age, tobacco smoking, high blood levels of certain lipids (lowdensity lipoprotein cholesterol, triglycerides) and low levels of high density lipoprotein (HDL) cholesterol, diabetes, high blood pressure, lack of physical activity and obesity, chronic kidney disease, excessive alcohol consumption, the use of illicit drugs (such as cocaine and amphetamines), and chronic high stress levels (Steptoe and Kivimäki, 2012). Epidemiology: Myocardial infarction is a common presentation of ischemic heart disease/coronary artery disease. The World Health Organization estimated that 12.2% of worldwide deaths were from ischemic heart disease; with it being the leading cause of death in high or middle income countries and second only to lower respiratory infections in lower income countries. Worldwide more than 3 million people have STEMIs and 4 million have NSTEMIs a year (White and Chew, 2008). Rates of death from ischemic heart disease have slowed or declined in most high income countries, although cardiovascular disease still accounted for 1 in 3 of all deaths in the USA in 2008. In contrast, ischemic heart disease is becoming a more common cause of death in the developing world. For example in India, ischemic heart disease had 5 Review of literature become the leading cause of death accounting for 1.46 million deaths (14% of total deaths) and deaths due to ischemic heart disease were expected to double during 1985–2015. Globally it is predicted that disability adjusted life years (DALYs) lost to ischemic heart disease will account for 5.5% of total DALYs in 2030, making it the second most important cause of disability (after unipolar depressive disorder), as well as the leading cause of death by this date (Roger et al., 2012). Heart attack rates are higher in association with intense exertion, (psychological stress or physical exertion), especially if the exertion is more intense than the individual usually performs.The period of intense exercise and subsequent recovery is associated with about a 6-fold higher myocardial infarction rate (compared with other more relaxed time frames) for people who are physically very fit for those in poor physical condition, the rate differential is over 35-fold higher (Saikku et al., 1992). There is an association of an increased incidence of a heart attack in the morning hours, more specifically around 9 a.m. Some investigators have noticed that the ability of platelets to aggregate varies according to a circadian rhythm, although they have not proven causation (Tofler et al., 1987). One observed mechanism for this phenomenon is increased pulse pressure, which increases stretching of the arterial walls this stretching results in significant shear stress on atheromas, which results in debris breaking loose from these deposits, This debris floats through the blood vessels, eventually clogging the major coronary arteries. Acute severe infection, such as pneumonia, can trigger myocardial infarction (Saikku et al., 1992). 6 Review of literature Myocardial infarction : Myocardial infarction (MI) or acute myocardial infarction (AMI), commonly known as a heart attack, results from the partial interruption of blood supply to a part of the heart muscle, causing the heart cells to be damaged or die. This is most commonly due to occlusion (blockage) of a coronary artery following the rupture of a vulnerable atherosclerotic plaque, which is an unstable collection of cholesterol and fatty acids and white blood cells in the wall of an artery. The resulting ischemia (restriction in blood supply) and ensuring oxygen shortage, if left untreated for a sufficient period of time, can cause damage or death (infarction) of heart muscle tissue (myocardium) (Mallinson, 2010). Classification: There are two basic types of acute myocardial infarction based on pathology: 1-Transmural: associated with atherosclerosis involving a major coronary artery. It can be subclassified into anterior, posterior, infe rior, lateral or septal. Transmural infarcts extend through the whole thickness of the heart muscle and are usually a result of complete occlusion of the area's blood supply In addition, on ECG, ST elevation and Q waves are seen. 2-Subendocardial: involving a small area in the subendocardial wall of the left ventricle, ventricular septum, or papillary muscles. The subendocardial area is particularly susceptible to ischemia (Reznik, 2010). Myocardial infarction can be further subclassified clinically into : 1- ST elevation MI (STEMI) 2- non-ST elevation MI (non-STEMI) 7 Review of literature based on ECG changes. The phrase heart attack is sometimes used incorrectly to describe sudden cardiac death which may or may not be the result of acute myocardial infarction. A heart attack is different from, but can be the cause of cardiac arrest, which is the stopping of the heartbeat, and cardiac arrhythmia, an abnormal heartbeat. It is also distinct from heart failure, in which the pumping action of the heart is impaired; however severe myocardial infarction may lead to heart failure (Van de Werf et al., 2008). A 2007 consensus document classifies myocardial infarction into five main types: Type 1: Spontaneous myocardial infarction related to ischemia due to a primary coronary event such as plaque erosion and/or rupture, fissuring, or dissection Type 2: Myocardial infarction secondary to ischemia due to either increased oxygen demand or decreased supply, e.g. coronary artery spasm, coronary embolism, anaemia, arrhythmias, hypertension, or hypotension Type 3 : Sudden unexpected cardiac death, including cardiac arrest, often with symptoms suggestive of myocardial ischaemia, accompanied by new ST elevation, or new LBBB, or evidence of fresh thrombus in a coronary artery by angiography and/or at autopsy, but death occurring before blood samples could be obtained, or at a time before the appearance of cardiac biomarkers in the blood Type 4 : Associated with coronary angioplasty or stents: Type 4a – Myocardial infarction associated with PCI Type 4b – Myocardial infarction associated with stent thrombosis as documented by angiography or at autopsy 8 Review of literature Type 5 : Myocardial infarction associated with CABG (Thygesen et al., 2007). Signs and symptoms A sizeable proportion of myocardial infarctions (22–64%) are "silent", that is without chest pain or other symptoms (Roe et al., 2010 ). The onset of symptoms in myocardial infarction (MI) is usually gradual, over several minutes, and rarely instantaneous. Chest pain is the most common symptom of acute myocardial infarction and is often described as a sensation of tightness, pressure, or squeezing. Chest pain due to ischemia (a lack of blood and hence oxygen supply) of the heart muscle is termed angina pectoris (Van de Werf et al., 2008). Pain radiates most often to the left arm, but may also radiate to the lower jaw, neck, right arm, back, and epigastrium, where it may mimic heartburn. Levine's sign, in which the patient localizes the chest pain by clenching their fist over the sternum, has classically been thought to be predictive of cardiac chest pain, although a prospective observational study showed that it had a poor positive predictive value (Marcus et al., 2007). Shortness of breath (dyspnea) occurs when the damage to the heart limits the output of the left ventricle, causing left ventricular failureand consequent pulmonary edema. Other symptoms include diaphoresis (an excessive from of sweating), weakness, light-headehness, nausea, vomiting, and palpitations. These symptoms are likely induced by a massive surge of catecholamines from thesympathetic nervous system which occurs in response to pain and the hemodynamic abnormalities that result from 9 cardiac dysfunction. Loss of Review of literature consciousness (due to inadequate cerebral perfusion and cardiogenic shock) and sudden death (frequently due to the development of ventricular fibrillation) can occur in myocardial infarctions (Van de Werf et al., 2008). Women and older patients report atypical symptoms more frequently than their male and younger counterparts. Women also report more numerous symptoms compared with men (2.6 on average vs 1.8 symptoms in men) (Canto et al., 2007). The most common symptoms of MI in women include dyspnea (shortness of breath), weakness, and fatigue. Fatigue, sleep disturbances, and dyspnea have been reported as frequently occurring symptoms that may manifest as long as one month before. the actual clinically manifested ischemic event. In women, chest pain may be less predictive of coronary ischemia than in men (McSweeney et al., 2003). At least one-fourth of all myocardial infarctions are silent, without chest pain or other symptoms. These cases can be discovered later on electrocardiograms, using blood enzyme tests or at autopsy without a prior history of related complaints. Estimates of the prevalence of silent myocardial infarctions vary between 22 and 64% (Valensi et al., 2011). A silent course is more common in the elderly, in patients with diabetes mellitus and after heart transplantation, probably because the donor heart is not fully innervated by the nervous system of the recipient, In people with diabetes, differences in pain threshold, autonomic neuropathy, and psychological factors have been cited as possible explanations for the lack of symptoms, any group of 10 Review of literature symptoms compatible with a sudden interruption of the blood flow to the heart are called an acute coronary syndrome (Davis et al., 2004). The differential diagnosis includes other catastrophic causes of chest pain, such as pulmonary embolism, aortic dissection, pericardial effusion causing cardiac and esophageal tamponade, rupture. Other tension non-catastrophic pneumothorax, differentials includegastroesophageal reflux and Tietze's syndrome (Boie , 2005). Risk factors for myocardial infarction : Myocardial infarction results from atherosclerosis. Smoking appears to be the cause of about 36% of coronary artery disease and obesity 20%. Lack of exercise has been linked to 7-12% of cases. Job stress appear to play a minor role accounting for about 3% of cases (Kivimäki et al., 2012). 1-Gender: men At any given age men are more at risk than women, particularly before menopause. but because in general women live longer than men ischemic heart disease causes slightly more total deaths in women (Graham et al., 2007). 2-Diabetes mellitus (type 1 or 2). 3-High blood pressure 4-Dyslipidemia/hypercholesterolemia: abnormal levels of lipoproteins in the blood, particularly high LDL-C, low HDL-C and high triglycerides. 5-Tobacco smoking, including secondhand smoke (Smith et al., 2006). 6-Short term exposure to air pollution including: carbon monoxide, nitrogen dioxide, and sulfur dioxide but not ozone. 11 Review of literature 7-Family history of ischaemic heart disease or myocardial infarction particularly if one has a first-degree relative (father, brother, mother, sister) who suffered a 'premature' myocardial infarction (defined as occurring at or younger than age 55 years (men) or 65 (women). 8-Obesity (defined by a body mass index of more than 30 kg/m², or alternatively by waist circumference or waist-hip ratio). 9-Lack of physical activity (Graham et al., 2007). 10-Psychosocial factors including, low socio-economic status, social isolation, negative emotions and stress increase the risk of myocardial infarction and are associated with worse outcomes after myocardial infarction. Socioeconomic factors such as a shorter education and lower income (particularly in women), and unmarried cohabitation are also correlated with a higher risk of MI (Nyboe et al., 1989) 11-Alcohol — Studies show that prolonged exposure to high quantities of alcohol can increase the risk of heart attack. 12-Oral contraceptive pill – women who use combined oral contraceptive pills have a modestly increased risk of myocardial infarction, especially in the presence of other risk factors, such as smoking. 13-Hyperhomocysteinemia (high homocysteine) in homocysteinuria is associated with premature atherosclerosis, whether elevated homocysteine in the normal range is causal is contentious (Clarke et al., 2011). 14-Calcium deposition is another part of atherosclerotic plaque formation. Calcium deposits in the coronary arteries can be detected with CT scans. Several studies have shown that coronary calcium can provide predictive information beyond that of classical risk factors (Detrano et al., 2008). 12 Review of literature Many of these risk factors are modifiable, so many heart attacks can be prevented by maintaining a healthier lifestyle. Physical activity, for example, is associated with a lower risk profile (Jensen ,et al. ,1991) Non-modifiable risk factors include age, sex, and family history of an early heart attack, which is thought of as reflecting a genetic predisposition.To understand epidemiological study results, it is important to note that many factors associated with MI mediate their risk via other factors. For example, the effect of education is partially based on its effect on income and marital status (Nyboe et al., 1989) Pathophysiology Acute myocardial infarction refers to two subtypes of acute coronary syndrome, namely non-ST-elevated myocardial infarction and ST-elevated myocardial infarction, which are most frequently (but not always) a manifestation of coronary artery disease (Moe and Wong, 2010). The most common triggering event is the disruption of an atherosclerotic plaque in an epicardial coronary artery, which leads to a clotting cascade, sometimes resulting in total occlusion of the artery (Dohi and Daida, 2010) Atherosclerosis is the gradual buildup of cholesterol and fibrous tissue in plaques in the wall of arteries (in this case, the coronary arteries), typically over decades. Blood stream column irregularities visible on angiography reflect artery lumen narrowing as a result of decades of advancing atherosclerosis (Spaan et al., 2008). 13 Review of literature Plaques can become unstable, rupture, and additionally promote a thrombus (blood clot) that occludes the artery; this can occur in minutes. When a severe enough plaque rupture occurs in the coronary vasculature, it leads to myocardial infarction (necrosis of downstream myocardium) (Dohi and Daida, 2010). If impaired blood flow to the heart lasts long enough, it triggers a process called the ischemic cascade; the heart cells in the territory of the occluded coronary artery die (chiefly through necrosis) and do not grow back. A collagen scar forms in its place. Recent studies indicate that another form of cell death called apoptosis also plays a role in the process of tissue damage subsequent to myocardial infarction (Krijnen et al.,2002). As a result, the patient's heart will be permanently damaged. This myocardial scarring also puts the patient at risk for potentially life threatening arrhythmias, and may result in the formation of a ventricular aneurysm that can rupture with catastrophic consequences. Injured heart tissue conducts electrical impulses more slowly than normal heart tissue. The difference in conduction velocity between injured and uninjured tissue can trigger re-entry or a feedback loop that is believed to be the cause of many lethal arrhythmias. The most serious of these arrhythmias is ventricular fibrillation (V-Fib/VF), an extremely fast and chaotic heart rhythm that is the leading cause of sudden cardiac death. Another life-threatening arrhythmia is ventricular tachycardia (VTach/VT), which may or may not cause sudden cardiac death. However, ventricular tachycardia usually results in rapid heart rates that prevent the heart from pumping blood effectively. Cardiac 14 output and blood Review of literature pressure may fall to dangerous levels, which can lead to further coronary ischemia and extension of the infarct (Dohi and Daida, 2010). Myocardial infarction diagnosis Medical societies recommend that the physician confirm that a patient is at high risk for myocardial infarction before conducting imaging tests to make a diagnosis. Patients who have a normal ECG and who are able to exercise, for example, do not merit routine imaging. Imaging tests such as stress radionuclide, myocardial, perfusion imaging or stress echocardiography can confirm a diagnosis when a patient's history, physical exam, ECG and cardiac biomarkers suggest the likelihood of a problem (Taylor et al., 2010). The diagnosis of myocardial infarction can be made after assessing patient's complaints and physical status. ECG changes, coronary angiogram and levels of cardiac markers help to confirm the diagnosis. ECG gives valuable clues to identify the site of myocardialdamage while coronary angiogram allows visualization of narrowing or obstructions in the heart vessels (Sudheer, 2011). A pathologist can diagnose a myocardial infarction based on anatomopathological findings. A chest radiograph and routine blood tests may indicate complications or precipitating causes and are often performed upon arrival to an emergency department. New regional wall motion abnormalities on an echocardiogram are also suggestive of a myocardial infarction (Skoufis and McGhie, 1998). 15 Review of literature WHO criteria. formulated in 1979 have classically been used to diagnose MI; a patient is diagnosed with myocardial infarction if two (probable) or three (definite) of the following criteria are satisfied: 1-Clinical history of ischaemic type chest pain lasting for more than 20 minutes 2-Changes in serial ECG tracings (Anonymous ,March 1979). 3-Rise and fall of serum cardiac biomarkers such as creatine kinaseMB fraction and troponin. The WHO criteria were refined in 2000 to give more prominence to cardiac biomarkers. cardiac troponin rise According to the accompanied by either new guidelines, typical a symptoms, pathological Q waves, ST elevation or depression, or coronary intervention is diagnostic of MI (Alpert et al., 2000). A number of diagnostic tests are available to detect heart muscle damage including, an electrocardiogram (ECG), echocardiography, cardiac MRI and various blood tests. The most often used blood markers are the creatine kinase-MB (CK-MB) fraction and the troponin levels (Roe et al., 2010). 16 Review of literature Laboratory tests: Types of cardiac markers include the following: Table (1): cardiac markers. Test Approximate Sensitivity and specificity peak Description Troponin is released during MI from the cytosolic pool of the myocytes. Its subsequent release is prolonged with degradation of actin The most sensitive and specific and myosin filaments. Isoforms of test the protein, T and I, are specific to for myocardial myocardium. Differential diagnosis damage. Because it has Troponin test increased compared MB, specificity with troponin superior 12 hours CKis marker of troponin elevation includes acute infarction, severe pulmonary embolism causing acute right heart a overload, heart failure, myocarditis. for Troponins can also calculate infarct myocardial injury. size but the peak must be measured in the 3rd day. After myocyte injury, troponin is released in 2–4 hours and persists for up to 7 days. CK-MB resides in the cytosol and facilitates movement of high energy Creatine Kinase MB) test It is relatively specific (CK- when skeletal muscle damage is not present. 10–24 hours phosphates into and out of mitochondria. It is distributed in a large number of tissues even in the skeletal muscle. Since it has a short duration, it cannot be used for late 17 Review of literature diagnosis of acute MI but can be used to suggest infarct extension if levels rise again. This is usually back to normal within 2–3 days. Lactate dehydrogenase catalyses the conversion of pyruvate to lactate. LDH-1 isozyme is normally found in the heart muscle and LDH-2 is found Lactate dehydrogenase (LDH) LDH is not as specific as troponin. predominately in blood serum. A 72 hours high LDH-1 level to LDH-2 suggest MI. LDH levels are also high in tissue breakdown hemolysis. It or can mean cancer, meningitis, encephalit is, or HIV. This is usually back to normal 10–14 days. Aspartate This was the first used. It is not transaminase( specific for heart damage, and it is AST) also one of the liver function tests. Myoglobin is used less than the other markers. Myoglobin is the Myoglobin (M b) low specificity for myocardial primary oxygen-carrying pigment 2 hours of muscle tissue. It is high when infarction muscle tissue is damaged but it lacks specificity. advantage 18 of It has responding the very Review of literature rapidly, rising and falling earlier than CK-MB or troponin. It also has been used in assessing reperfusion after thrombolysis. IMA can be detected via the albumin cobalt binding (ACB) test, a limited available FDA approved assay. Myocardial ischemia alters the N-terminus of albumin reducing the ability of cobalt to bind to albumin. IMA measures ischemia Ischemiamodified in the blood vessels and thus low specificity returns results in minutes rather albumin (IMA) than traditional markers of necrosis that take hours. ACB test has low specificity therefore generating high number of false positives and must be used in conjunction with typical acute approaches such as ECG and physical exam. Additional studies are required. This is increased in patients with heart failure. It has been approved Pro-brain as a marker for acute congestive natriuretic heart failure. Pt with < 80 have a peptide much higher rate of symptom free survival within a year. Generally, pt 19 Review of literature with CHF will have > 100. Glycogen phosphorylase isoenzyme BB (abbreviation: GPBB) is an isoenzyme of phosphorylase. glycogen Glycogen phosphorylase exists in 3 isoforms. One of these Isoforms is GP-BB. This isoform exists in heart and brain tissue. Because of the blood– brain barrier GP-BB can be seen as heart muscle specific. During the Glycogen phosphorylase process of ischemia, GP-BB is 0.854 and 0.767 7 hours converted into a soluble form and is isoenzyme BB released into the blood. This isoform of the enzyme exists in cardiac (heart) and brain tissue. GPBB is one of the "new cardiac markers" which are discussed to improve early diagnosis in acute coronary syndrome. A rapid rise in blood levels can be seen in myocardial infarction and unstable angina. GP-BB elevated 1–3 hours after process of ischemia. (Lewis et al ., 2008 and Lippi et al.,2013) 20 Review of literature Inflammation is known to be an important step in the process of atherosclerotic plaque formation. C-reactive protein (CRP) is a sensitive but non-specific marker forinflammation. Elevated CRP blood levels, especially measured with high-sensitivity assays, can predict the risk of MI, as well as stroke and development of diabetes. Moreover, some drugs for MI might also reduce CRP levels (Wilson et al., 2006). Prevention The risk of a recurrent myocardial infarction decreases with strict blood pressure management and lifestyle changes, chiefly smoking cessation, regular exercise, a sensible diet for those with heart disease, and limitation of alcohol intake. People are usually commenced on several long-term medications post-MI, with the aim of preventing secondary cardiovascular events such as further myocardial infarctions, congestive heart failure or cerebrovascular accident (CVA) (Rossi et al., 2006). Unless contraindicated, such medications may include: - Antiplatelet drug therapy such as aspirin and/or clopidogrel should be continued to reduce the risk of plaque rupture and recurrent myocardial infarction. Aspirin is first-line, owing to its low cost and comparable efficacy, with clopidogrel reserved for patients intolerant of aspirin. The combination of clopidogrel and aspirin may further reduce risk of cardiovascular events, however the risk of hemorrhage is increased (Peters et al., 2003). - Beta blocker therapy such as metoprolol or carvedilol should be commenced. These have been particularly beneficial in high-risk patients such as those with left ventriculardysfunction and/or continuing cardiac ischaemia. β-Blockers decrease mortality and 21 Review of literature morbidity. They also improve symptoms of cardiac ischemia in NSTEMI (Dargie ,2001) Treatment : Immediate treatment for suspected acute myocardial infarction includes oxygen, aspirin, and sublingualnitroglycerin, most cases of myocardial infarction with ST elevation on ECG (STEMI) are treated with reperfusion therapy, such as percutaneous coronary intervention (PCI) or thrombolysis (Roe et al.,2010). Non-ST elevation myocardial infarction (NSTEMI) may be managed with medication, although PCI may be required if the patient's risk warrants it. People who have multiple blockages of their coronary arteries, particularly if they also have diabetes mellitus, may benefit from bypass surgery (CABG) (Hamm et al., 2011). The European Society of Cardiology guidelines in 2011 proposed treating the blockage causing the myocardial infarction by PCI and performing CABG later when the patient is more stable. Rarely CABG may be preferred in the acute phase of myocardial infarction, for example when PCI has failed or is contraindicated (Van de Werf et al , 2008). ACE inhibitor therapy should be commenced 24–48 hours post-MI in hemodynamically stable patients, particularly in patients with a history of MI, diabetes mellitus,hypertension, anterior location of infarct (as assessed by ECG), and/or evidence of left ventricular dysfunction. ACE inhibitors reduce mortality, the development of heart failure, and decrease ventricular remodelling post-MI (Pfeffer et al.,1992). 22 Review of literature Statin therapy has been shown to reduce mortality and morbidity post-MI. The effects of statins may be more than their LDL lowering effects. The general consensus is that statins have plaque stabilization and multiple other ("pleiotropic") effects that may prevent myocardial infarction in addition to their effects on blood lipids (Ray and Cannon, 2005). The aldosterone antagonist agent eplerenone has been shown to further reduce risk of cardiovascular death post-MI in patients with heart failure and left ventricular dysfunction, when used in conjunction with standard therapies above. Spironolactone is another option that is sometimes preferable to eplerenone due to cost. Evidence supports the consumption of polyunsaturated fats instead of saturated fats as a measure of decreasing coronary heart disease (Mozaffarian et al., 2010). In high-risk people there is no clear-cut decrease in potentially fatal arrhythmias due to omega-3 fatty acids. And they may increase risk in some groups. Giving heparin to people with heart conditions like unstable angina and some forms of heart attacks reduces the risk of having another heart attack. However, heparin also increases the chance of minor bleeding (Magee et al., 2008). Angina pectoris : (Tobin., 2010). Angina pectoris – commonly known as angina – is chest pain due to ischemia of the heart muscle, generally due to obstruction or spasm of the coronary arteries, the main cause of Angina pectoris is coronary artery disease, due to atherosclerosis of the arteries feeding the heart. The term derives from the Latin angina ("infection of the throat") from the Greek ἀγχόνη ankhonē ("strangling"), and the Latin pectus ("chest"), and can therefore be translated as "a strangling feeling in the chest". 23 Review of literature There is a weak relationship between severity of pain and degree of oxygen deprivation in the heart muscle (i.e., there can be severe pain with little or no risk of a Myocardial infarction (commonly known as a heart attack), and a heart attack can occur without pain). In some cases Angina can be extremely serious and has been known to cause death. People that suffer from average to severe cases of Angina have an increased percentage of death before the age of 55, usually around 60%. Worsening ("crescendo") angina attacks, sudden-onset angina at rest, and angina lasting more than 15 minutes are symptoms ofunstable angina (usually grouped with similar conditions as the acute coronary syndrome). As these may herald myocardial infarction (a heart attack), they require urgent medical attention and are generally treated as a presumed heart attack. Classification of angina : (Tobin., 2010) 1- Stable angina Also known as effort angina, this refers to the more common understanding of angina related to myocardial ischemia. Typical presentations of stable angina is that of chest discomfort and associated symptoms - Precipitated by some activity (running, walking, etc.) - With minimal or non-existent symptoms at rest or with administration of sublingual nitroglycerin. - -Symptoms typically abate several minutes following cessation of precipitating activities. - Reoccur when activity resumes. In this way, stable angina may be thought of as being similar to intermittent claudication symptoms. 24 Review of literature 2- Unstable angina Unstable angina (UA) (also "crescendo angina;" this is a form of acute coronary syndrome) is defined as angina pectoris that changes or worsens (Simons and Michael, 2000). It has at least one of these three features: -It occurs at rest (or with minimal exertion), usually lasting >10 min; -It is severe and of new onset (i.e., within the prior 4–6 weeks); and/or -It occurs with a crescendo pattern (i.e., distinctly more severe, prolonged, or frequent than before). UA may occur unpredictably at rest which may be a serious indicator of an impending heart attack. What differentiates stable angina from unstable angina (other than symptoms) is the pathophysiology of the atherosclerosis. The pathophysiology of unstable angina is the reduction of coronary flow due to transient platelet aggregation on apparently normal endothelium, coronary artery spasms or coronary thrombosis (Mosca et al., 2011). 3- Microvascular angina Microvascular Angina or Angina Syndrome X is characterized by angina-like chest pain, but the cause is different. The cause of Microvascular Angina is unknown, but it appears to be the result of spasm in the tiny blood vessels of the heart, arms and legs. Since microvascular angina isn't characterized by arterial blockages, it's harder to recognize and diagnose, but its prognosis is excellent (Guyton and Arthur, 2006). 25 Review of literature Major risk factors : (Moyer and Virginia .,2012). 1-Age (≥ 55 years for men, ≥ 65 for women) 2-Cigarette smoking 3-Diabetes mellitus (DM) 4-Dyslipidemia 5-Family history of premature cardiovascular disease (men <55 years, female <65 years old) 6-Hypertension (HTN) 7-Kidney disease (microalbuminuria or GFR<60 mL/min) 8-Obesity (BMI ≥ 30 kg/m2) 9-Physical inactivity 10-Prolonged psychosocial stress. Routine counselling of adults to advise them to improve their diet and increase their physical activity has not been found to significantly alter behaviour, and thus is recommended. Pathophysiology The process starts with atherosclerosis, and when inflamed leads to an active plaque, which undergoes thrombosis and results in acute ischemia, which finally results in cell necrosis after calcium entry. Studies show that 64% of all unstable anginas occur between 10 PM and 8 AM when patients are at rest (Simons and Michael ,2000). In stable angina, the developing atheroma is protected with a fibrous cap. This cap (atherosclerotic plaque) may rupture in unstable angina, allowing blood clots to precipitate and further decrease the lumen of the coronary vessel. This explains why an unstable angina appears to be independent of activity (Mosca et al., 2011). 26 Review of literature Angina results when there is an imbalance between the heart's oxygen demand and supply. This imbalance can result from an increase in demand (e.g. during exercise) without a proportional increase in supply (e.g. due to obstruction or atherosclerosis of the coronary arteries). However, the pathophysiology of angina in females varies significantly as compared to males. Non-obstructive coronary disease is more common in females (Vaccarino, 2010). Signs and symptoms : (Sun et al., 2002) Angina pectoris can be quite painful, but many patients with angina complain of chest discomfort rather than actual pain: the discomfort is usually described as a pressure, heaviness, tightness, squeezing, burning, or choking sensation. Apart from chest discomfort, anginal pains may also be experienced in the epigastrium (upper central abdomen), back, neck area, jaw, or shoulders. This is explained by the concept of referred pain, and is due to the spinal level that receives visceral sensation from the heart simultaneously receiving cutaneous sensation from parts of the skin specified by that spinal nerve's dermatome, without an ability to discriminate the two. Typical locations for referred pain are arms (often inner left arm), shoulders, and neck into the jaw. Angina is typically precipitated by exertion or emotional stress. It is exacerbated by having a full stomach and by cold temperatures. Pain may be accompanied by breathlessness, sweating and nausea in some cases. In this case, the pulse rate and the blood pressure increases. Chest pain lasting only a few seconds is normally not angina (such as Precordial catch syndrome). 27 Review of literature Myocardial ischemia comes about when the myocardia (the heart muscles) receive insufficient blood and oxygen to function normally either because of increased oxygen demand by the myocardia or by decreased supply to the myocardia. This inadequate perfusion of blood and the resulting reduced delivery of oxygen and nutrients is directly correlated to blocked or narrowed blood vessels. Some experience "autonomic symptoms" (related to increased activity of the autonomic as nausea, vomiting and pallor. include cigarette pressure, sedentary nervous Major risk smoking, diabetes, high lifestyle and family system) factors such for angina cholesterol, high blood history of premature heart disease. A variant form of angina (Prinzmetal's angina) occurs in patients with normal coronary arteries or insignificant atherosclerosis. It is thought to be caused by spasms of the artery. It occurs more in younger women. One study found that smokers with coronary artery disease had a significantly increased level of sympathetic nerve activity when compared to those without. This is in addition to increases in blood pressure, heart rate and peripheral vascular resistance associated with nicotine which may lead to recurrent angina attacks. Additionally, CDC reports that the risk of CHD (Coronary heart disease), stroke, and PVD (Peripheral vascular disease) is reduced within 1–2 years of smoking cessation. In another study, it was found that after one year, the prevalence of angina in smoking men under 60 after an initial attack was 40% less in those who had quit smoking compared to those who continued. Studies have found that there are short term and long term benefits to smoking cessation. (Shinozaki et al., 2008). 28 Review of literature Myocardial ischemia can result from: (Podrid, 2012) A reduction of blood flow to the heart that can be caused by stenosis, spasm, or acute occlusion (by an embolus) of the heart's arteries. Resistance of the blood vessels. This can be caused by narrowing of the blood vessels; a decrease in radius. Blood flow is proportional to the radius of the artery to the fourth power. Reduced oxygen-carrying capacity of the blood, due to several factors such as a decrease in oxygen tension and hemoglobin concentration. This decreases the ability of hemoglobin to carry oxygen to myocardial tissue. Atherosclerosis is the most common cause of stenosis (narrowing of the blood vessels) of the heart's arteries and, hence, angina pectoris. Some people with chest pain have normal or minimal narrowing of heart arteries; in these patients, vasospasm is a more likely cause for the pain, sometimes in the context of Prinzmetal's angina and syndrome X. Myocardial ischemia also can be the result of factors affecting blood composition, such as reduced oxygen-carrying capacity of blood, as seen with severe anemia (low number of red blood cells), or longterm smoking (Vaccarino, 2010). Diagnosis : (Banks, et al., 2010) Suspect angina in people presenting with tight, dull, or heavy chest discomfort which is: - Retrosternal or left-sided, radiating to the left arm, neck, jaw, or back. Associated with exertion or emotional stress and relieved 29 Review of literature within several minutes by rest. Precipitated by cold weather or a meal. - Some people present with atypical symptoms, including breathlessness, nausea, or epigastric discomfort or burping. These atypical symptoms are particularly likely in older people, women, and those with diabetes. - Angina pain is not usually sharp or stabbing or influenced by respiration. Anti-acids and simple analgesia do not usually relieve the pain. If chest discomfort (of whatever site) is precipitated by exertion, relieved by rest, and relieved by glyceryl trinitrate, the likelihood of angina is increased. Laboratory tests : (Fox K, 2010) Recommendations for laboratory investigation in initial assessment of stable angina. Class I (in all patients) Fasting lipid profile, including TC, LDL, HDL, and triglycerides (level of vidence B). 1. Fasting glucose (level of evidence B) 2. Full blood count including Hb and white cell count (level of evidence B) 3. Creatinine (level of evidence C) Class I (if specifically indicated on the basis of clinical evaluation) 1- markers of myocardial damage if evaluation suggests clinical instability or ACS (level of evidence A). 2- Thyroid function if clinically indicated (level of evidence C). 30 Review of literature Class IIa (1) Oral glucose tolerance test (level of evidence B) Class IIb (1) Hs-C-reactive protein (level of evidence B) (2) Lipoprotein(a), ApoA, and ApoB (level of evidence B) (3) Homocysteine (level of evidence B) (4) HbA1c (level of evidence B) (5) NT-BNP (level of evidence B) Recommendations for blood tests for routine reassessment in patients with chronic stable angina. Class IIa (1) Fasting lipid profile and fasting glucose on an annual basis (level of evidence C). N.B Class Level of symptoms Class I ‘Ordinary activity does not cause angina’ Angina with strenuous or rapid or prolonged exertion only Class II ‘Slight limitation of ordinary activity’ Angina on walking or climbing stairs rapidly, walking uphill or exertion after meals, in cold weather, when under emotional stress, or only during the first few hours after awakening. Class III ‘Marked limitation of ordinary physical activity’ Angina on walking one or two blocksa on the level or one flight of stairs at a normal pace under normal conditions. Class IV ‘Inability to carry out any physical activity without discomfort’ or ‘angina at rest’. 31 Review of literature In angina patients who are momentarily not feeling any one chest pain, an electrocardiogram (ECG) is typically normal, unless there have been other cardiac problems in the past. During periods of pain, depression or elevation of the ST segment may be observed. To elicit these changes, an exercise ECG test ("treadmill test") may be performed, during which the patient exercises to their maximum ability before fatigue, breathlessness or, importantly, pain intervenes; if characteristic ECG changes are documented (typically more than 1 mm of flat or downsloping ST depression), the test is considered diagnostic for angina. Even constant monitoring of the blood pressure and the pulse rate can lead us to some conclusion regarding the angina. The exercise test is also useful in looking for other markers of myocardial ischaemia. (Podrid, 2012). In patients in whom such noninvasive testing is diagnostic, a coronary angiogram is typically performed to identify the nature of the coronary lesion, and whether this would be a candidate for angioplasty, coronary artery bypass graft (CABG), treatment only with medication, or other treatments. There has been research which concludes that a frequency is attained when there is increase in the blood pressure and the pulse rate. This frequency varies normally but the range is 45–50 kHz for the cardiac arrest or for the heart failure In patients who are in hospital with unstable angina (or the newer term of "high risk acute coronary syndromes"), those with resting ischaemic ECG changes or those with raised cardiac enzymes such as troponin may undergo coronary angiography directly. (Banks et al., 2010). 32 Review of literature Therefore, there is need for identifying new biomarkers, which alone or in combination with other risk markers are useful in monitoring treatment and as prognostic markers for future cardiovascular events in patients with ischemic heart disease. (Camilla and Henrik, 2009). 33 Review of literature YKL-40 The inflammatory biomarker YKL-40: (Camilla and Henrik, 2009). It has been found elevated in patients with both acute and stable chronic cardiovascular diseases. Therefore, YKL-40 could potentially be a new useful biomarker of disease severity, prognosis and survival in patients with ischemic heart disease . YKL-40 is an inflammatory glycoprotein involved in endothelial dysfunction by promoting chemotaxis, cell attachment and migration, and tissue remodelling as a response to endothelial damage. YKL-40 protein expression is seen in macrophages and smooth muscle cells in atherosclerotic plaques with the highest expression seen in macrophages in the early lesion of atherosclerosis. Several studies demonstrate, that elevated serum YKL-levels are independently associated with the presence and extent of coronary artery disease and even higher YKL-40 levels are documented in patients with myocardial infarction. Moreover, elevated serum YKL-40 levels have also been found to be associated with all-cause as well as cardiovascular mortality . YKL-40 levels are elevated both in patients with type 1 and type 2 diabetes, known to be at high risk for the development of cardiovascular diseases, when compared to non-diabetic persons. A positive association between elevated circulating YKL-40 levels and increasing levels of albuminuria have been described in patients with type 1 diabetes indicating a role of YKL-40 in the progressing vascular damage resulting in microvascular disease. There is a relatin between YKL-40 and 34 Review of literature endothelial dysfunction, atherosclerosis, cardiovascular disease and diabetes and look ahead on future perspectives of YKL-40 research. YKL-40 - biology and physiology: YKL-40 is a 40 kDa heparin- and chitin-binding glycoprotein also known as human cartilage glycoprotein 39 (HC-gp39) 38-kDa heparinbinding glycoprotein or chitinase-3-like protein 1 (CHI3L1) The abbreviation YKL-40 is based on the one letter code for the first three Nterminal amino acids, tyrosine (Y), lysine (K) and leucine (L) and the apparent molecular weight of YKL-40 (Camilla and Henrik 2009). The CHI3L1 gene for human YKL-40 is localized in a highly conserved area on chromosome 1q31-q32 and the crystal structure of YKL-40 has been described . YKL-40 belongs to the family 18 of glycosyl hydrolases comprising chitinases from various species, but YKL-40 is without any enzymatic properties (Johansen, 2006). YKL-40 is secreted by various cell-types including macrophages, chondrocytes and some types of cancer cells. YKL-40 lacks chitinase activity due to mutations within the active site (conserved sequence: DXXDXDXE ; YKL-40 sequence: DGLDLAWL). The exact physiological role of YKL-40 is not known, but it has been implicated in development, inflammatory disease (such as asthma, and cancer progression) (Ober, et al., 2008). Function Chitinases catalyze the hydrolysis of chitin, which is an abundant glycopolymer found in insect exoskeletons and fungal cell walls. The glycoside hydrolase 18 family of chitinases includes eight human family members. This gene encodes a glycoprotein member of the glycosyl 35 Review of literature hydrolase 18 family. The protein lacks chitinase activity and is secreted by activated macrophages, chondrocytes, neutrophils and synovial cells. The protein is thought to play a role in the process of inflammation and tissue remodeling (Francescone et al., 2011). YKL-40 is secreted in vitro by a variety of cells and seems especially involved in activation of the innate immune system and in cell processes in relation to extracellular matrix remodelling .YKL-40 induce the maturation of monocytes to macrophages, and is secreted by macrophages during late stages of differentiation and by activated macrophages (Rehli ,et al 2003). Studies show that the differentiation and maturation of CD14+ monocytes to CD14-, CD16+ macrophages are attended by an expression of YKL-40 from CD16+ macrophages .YKL-40 has also been shown to be an adhesion and migration factor for vascular cells and is secreted by differentiated vascular smooth muscle cells (VSMCs) (Nishikawa and Millis 2003). The knowledge about the physiological function and the mechanisms by which YKL-40 mediates its effects is still scarce. Immunohistochemical studies of different types of normal human tissues show, that cells with a high cellular activity, e.g. a high level of metabolic activity and/or proliferation, have especially high YKL-40 expression (Johansen et al., 2007). In vivo YKL-40 protein expression is found in human VSMCs in adventitial vessels and in subpopulations of macrophages and VSMCs in different tissues with inflammation and extracellular matrix remodelling as in atherosclerotic plaques (Nishikawa and Millis, 2003). 36 Review of literature YKL-40 mRNA and protein expression are found in tissues from all germ layers and are present during the early development of the human musculoskeletal system where they seem associated with cell proliferation, differentiation and tissue morphogenesis (Johansen et al., 2007). Other studies show, that YKL-40 stimulates the proliferation of human connective tissue cells (fibroblasts, chondrocytes, synovial cells) in a dose-dependent manner in a functional concentration range similar to that of insulin-like growth factor (IGF-1). When present in suboptimal concentrations, YKL-40 and IGF-1 work in a synergistic fashion (Recklies et al., 2002). In mouse studies, YKL-40 stimulates the antigen-induced T-helper 2-response and seems to induce tissue inflammation and fibrosis mediated by IL-13. In this sense, YKL-40 plays an essential role in antigen sensitization and IgE induction as well as in activation of innate immune cells (Lee et al., 2009). In fibroblasts and synovial cells YKL-40 mediates a mitogenic effect through initiation of mitogen-activated protein kinase (MAPK) and phosphoinoside-3 kinase (PI3K) signalling pathways by phosphorylation of the extracellular signal-regulated kinase-1 and 2 (ERK1/ERK2) and protein kinase B (AKT), respectively. Both pathways are required for the cells to complete mitosis and the activation of these pathways stimulates the growth of connective tissue cells (Recklies et al., 2002). In fibroblasts and chondrocytes YKL-40 reduces the activation of p38 and SAPK/JNK MAPKs which counteracts the inflammatory responses to TNFα and IL-1. This leads to reduced concentrations of 37 Review of literature matrix metalloproteinases (MMPs) and IL-8. The modulation of p38 and SAPK/JNK by YKL-40 is mediated through the PI3K and the induction and continued secretion of YKL-40 require sustained activation of NfκB. (Ling et al., 2005). YKL-40 has no effect on the signalling pathways p38 and SAPK/JNK MAPKs when present without the presence of TNFα and IL1 and similar do not affect the MMP or IL-8 production. This suggests that YKL-40 expression is an anti-inflammatory counteract of the inflammatory response mediated by TNFα and IL-1 beside its apparent function as a growth factor (Recklies et al., 2002). The activation of cytoplasmatic signal-transduction pathways suggests, that YKL-40 interacts with one or several signalling components on the plasma membrane. However, specific cell surface receptors or potential YKL-40 ligands remain to be determined. No difference in serum or plasma YKL-40 levels has been found between genders (Johansen 2006). YKL-40 in endothelial dysfunction and atherosclerosis: (Malinda et al., 2009). The participation of YKL-40 in inflammatory states and vascular processes implies that YKL-40 may play a role in endothelial dysfunction and atherosclerosis. In endothelial dysfunction, elevated YKL-40 levels seem to be involved in relation to cell migration, reorganization and tissue remodelling as a response to endothelial damage. In vitro VSMCs from explants of swine thoracic aorta syntesize YKL-40 during the time of transition from monolayer culture to a nonproliferating differentiated multilayer culture .The YKL-40 secretion 38 Review of literature continues during the reorganisation of the cells where multicellular nodules are formed. In these nodules the cells re-express markers of differentiated VSMCs. This in vitronodule forming process mimics some of the characteristics of the in vivo changes that occur in VSMCs following injury, where media smooth muscle cells differentiate, migrate and contribute to the process of restenosis and neointima formation. In vitro studies also show that YKL-40 promotes chemotaxis, cell attachment, spreading and migration of vascular endothelial cells which suggest a role of YKL-40 in the atherosclerotic plaque formation, where smooth muscle cells are induced to migrate through the intima in response to exogenous signals. YKL-40 also modulates vascular endothelial cell morphology by promoting the formation of branching tubules, indicating that YKL-40 has a role in angiogenesis by stimulating the migration and reorganization of VSMCs .These in vitro studies are supported by immunohistochemical analysis which has shown in vivo protein expression of YKL-40 in human smooth muscle cells in atherosclerotic plaques . YKL-40 mRNA expression is highly up-regulated in distinct subsets of macrophages in the atherosclerotic plaque, a plaque that is characterized by the infiltration of monocytes into the subendothelial space of the vessel wall and a subsequent lipid accumulation in the activated macrophages. Particularly macrophages that infiltrate deeper in the lesion show high YKL-40 mRNA expression and the highest expression is seen in macrophages in the early lesion of atherosclerosis (Boot et al., 1999). 39 Review of literature An in vitro study with emphasis on biomarker discovery for atherosclerosis by proteomics, show elevated YKL-40 levels in the supernatant of macrophages following treatment with oxidized lowdensity lipoprotein, a process that mimics the formation of "foam cells (Fach et al., 2004). This also suggests a role of YKL-40 in the differentiation of monocytes to lipid-laden macrophages during formation of the atherosclerotic plaque. YKL-40 in cardiovascular disease: (Kastrup et al., 2009). In the last few years, several clinical studies have described elevated YKL-40 levels in several cardiovascular conditions as well as described an association between YKL-40 and mortality. Studies show, that elevated YKL-levels are independently associated with the presence of CAD. One study even found, that YKL-40 levels increase with the extent of CAD defined by the number of stenosed vessels as assessed by coronary angiography .This findings indicate, that plasma YKL-40 levels could be a quantitative indicator of disease progression as well as of disease presence. In patients suffering myocardial infarction (MI) even higher YKL-levels have been documented, and YKL-levels remain higher in patients with prior MI compaired to individuals without previous MI. There seems to be no difference in YKL-40 levels between MI patients with or without ST elevations, but higher YKL-40 levels were seen in thrombolyzed patients compared with non-thrombolyzed patients during the first 24 hours after the event, indicating that YKL-40 is 40 Review of literature released from the dissolved thrombosis. Elevated YKL-40 levels have also been documented in individuals with atrial fibrillation (AF) where the highest YKL-40 levels were found in patients with permanent AF compared to patients with persistent AF suggesting an association between the chronicity of AF and the inflammatory burden (Nojgaard et al., 2008). Elevated YKL-40 levels have also been found to be associated with all-cause as well as cardiovascular mortality in patients with stable CAD .Furthermore, increasing mortality rates with increasing YKL-40 levels at baseline are also seen over a 5 year period in the general population above 50 years of age without known diabetes or CVD in which YKL-40 were also found to be an independent predictor of overall as well as of cardiovascular mortality (Kastrup et al., 2009). YKL-40 and diabetes It has been demonstrated, that patients with type 1 diabetes as well as patients with type 2 diabetes have elevated plasma YKL-40 levels . In type 2 diabetes patients YKL-40 levels are correlated with insulin resistance ,and in a single study also with the diabetic lipid profile . Some studies have also shown a correlation between YKL-40 and glycemic parameters such as hemoglobin A1c and fasting glucose whereas others have not (Rathcke et al., 2006). Individuals with diabetes have in general a 2- to 4-fold increased risk of subsequent CVD .Persistent microalbuminuria is associated with an increased risk of CVD in both patients with type 1 and type 2 diabetes . Patients with type 1 diabetes have up to a 9-fold increased mortality risk from ischemic heart disease, excessively higher in patients under 30 years of age (Rossing et al., 2005). 41 Review of literature In patients with type 1 diabetes a positive association between elevated plasma YKL-40 levels and increasing levels of albuminuria has been described .This finding indicates a role of YKL-40 in the progressing vascular damage in the kidneys resulting in complicating microvascular disease. This hypothesis is supported by the finding that YKL-40 and urinary albumin/creatinine ratio (UACR) are independent markers with only weak intercorrelation that seem to predict overall as well as cardiovascular mortality in a synergistic way in the general population above 50 years of age without known diabetes or CVD over a 5 year period (Rathcke et al., 2009). YKL-40 in other clinical conditions The exact biological function of YKL-40 in cancer is unknown, but YKL-40 seems to play an important role in tumor invasion. The signalling pathways MAPK/ERK1/2 and PI3K/AKT which YKL-40 has been demonstrated to mediate its effects through in other conditions ,are critical in the malignant phenotype of glioblastoma and have been shown to govern proliferation and survival, invasiveness and radiation resistance .Furthermore, activation of the PI3K/AKT-pathway is correlated with increased tumor grade, lesser likelihood of apoptosis and decreased overall survival . However, the functional ligand for the chitinbinding site in YKL-40 in relation to cancer is not presently known (Pelloski et al., 2006). Serum YKL-40 levels have been found to be elevated in other clinical conditions not directly related to atherosclerosis or cardiovascular disease. Several studies describe elevated YKL-40 levels in patients with different types of cancer .YKL-40 levels seem to be related to tumor grade and burden, short recurrence-free interval and short disease-free and overall survival (Johansen, 2006). 42 Review of literature An in vitro study has shown, that ectopic expression of YKL-40 in breast and colon cancer cells respectively led to tumor formation with an extensive angiogenic phenotype and that recombinant YKL-40 protein promoted vascular endothelial cell angiogenesis whereas blockade of YKL-40 suppressed tumor angiogenesis both in vitro and in vivo Furthermore, immunohistochemical analysis of human breast cancer showed a correlation between YKL-40 expression and blood vessel density (Pelloski et al., 2006). Therefore, the occurrence of high YKL-40 levels in highly differentiated and advanced cancers and recurrent cancer states could be explained by the role of YKL-40 in both angiogenesis and fibrogenesis, since highly differentiated tumours are characterized by high vascularization and a high turnover of extracellular matrix. YKL-40 is not tumor specific and the studies of YKL-40 as a screening marker for cancer and as a marker useful for monitoring therapeutic results differ. Furthermore, YKL-40 seems not suited as a tumor marker due to low specificity and sensitivity (Johansen et al., 2006). Role in cancer YKL-40 is cytokine that the tumor micro-environment is and present in the at high serum levels of in cancer patients. Elevated levels of YKL-40 correlate strongly with stage and outcome of various types of cancer, which establish YKL-40 as a biomarker of disease severity. Targeting YKL-40 with neutralizing antibodies has been proven effective as a treatment in animal models of glioblastoma multiforme. YKL-40 is able to promote angiogenesis through VEGF-dependent and independent pathways. 43 Review of literature YKL-40 also enhances tumor survival in response to gammairradiation (Francescone et al., 2011). Methods for determination of YKL-40 in tissues and body fluids: (Johansen, 2006). Microarray cDNA analysis. In situ hybridization. Immunohistochemical analysis. Radio-and enzyme-linked immunoassays for the determination of YKL-40. 44 Review of literature Myeloperoxidase Myeloperoxidase (MPO) is an enzyme stored in azurophilic granules of polymorphonuclear neutrophils and macrophages and released into extracellular fluid in the setting of inflammatory process. The observation that myeloperoxidase is involved in oxidative stress and inflammation has been a leading factor to study myeloperoxidase as a possible marker of plaque instability and a useful clinical tool in the evaluation of patients with coronaryheart disease. Myeloperoxidase (MPO) is a well-known enzyme, mainly released by activated neutrophils, characterised by powerful pro-oxidative and proinflammatory properties. Myeloperoxidase has been proposed as a useful risk marker and diagnostic tool in acute coronary syndromes and in patients admitted to emergency room for chest pain (Valentina et al., 2008). Pathophysiological Role of Myeloperoxidase in Ischemic Heart Disease: Myeloperoxidase (MPO) was traditionally considered to be a bactericidal agent. Recent investigations revealed a crucial role of MPO in chronic, nonmicrobial inflammatory processes such as neurodegenerative diseases and atherosclerosis. MPO, a glycosylated, arginine-rich, extremely basic protein (isoelectric point >10) is comprised of two subunits, encoded within a single mRNA. Two of each subunits are assembled with heme molecules to produce the functional enzyme (donor: hydrogen peroxide, oxidoreductase, (Madlusudhana, et al., 20l1). 45 EC 1.11.1.7) Review of literature Oxidative stress and inflammation play important roles in the destabilization of coronary artery disease (CAD) leading pathogenesis of to acute coronary syndromes (ACS). Infiltrating macrophages and neutrophils participate in the transformation of stable coronary artery plaques to unstable lesions . There has been a renewed interest in MPO, a proinflammatory enzyme that is abundant in ruptured plaque and can be measured in peripheral blood (Takahiko et al., 2002). MPO is stored in primary azurophilic granules of leukocytes and the enzyme accounts for up to 5 and 1% of total cell protein content, in neutrophilic polymorphonuclear leukocytes (neutrophils) and monocytes, respectively.The ability of MPO to generate hypochlorous acid/hypochlorite (HOCl/OCl−) from hydrogen peroxide in the presence of chloride ions is a unique and defining activity for this enzyme. The importance of MPO-catalyzed oxidative reactions and formation of a variety of chlorinated protein and lipid adducts (with hypochlorous acid as the major oxidant in causing tissue injury by phagocytic cells) has been emphasized. (Madlusudhana, et al., 20l1). MPO catalyzes the conversion of chloride and hydrogen peroxide to hypochlorite and is secreted during inflammatory condition. It has been implicated in the oxidation of lipids contained within LDL cholesterol. In addition, MPO consumes endothelial-derived NO, thereby reducing NO bioavailability and impairing its vasodilating and anti-inflammatory properties. Major evidence for MPO as enzymatic catalyst for oxidative modification of lipoproteins in the artery wall has been suggested in a number of studies performed with low-density lipoprotein (Holvoet, 1998). 46 Review of literature Furthermore, high levels of MPO-mediated endothelial dysfunction may be an important mechanistic link between oxidation, inflammation, and cardiovascular disease (CVD) .An elevated level of plasma MPO served as independent predictor of increased risk of myocardial infarction . However, the role of MPO in chronic kidney disease (CKD) is poorly understood, and not much data is available regarding the variations of this enzyme in these patients. It was speculated that raised levels of this enzyme might be one of the factors responsible for the increased risk that these patients have for developing CVD (Madlusudhana, et al., 20l1). In contrast to low-density lipoprotein, plasma levels of highdensity lipoprotein (HDL)-cholesterol and apoAI, the major apolipoprotein of HDL, inversely correlate with the risk of developing coronary artery disease. There is now strong evidence that HDL is a selective in vivo target for MPO-catalyzed oxidation, that may represent a specific molecular mechanism for converting the cardioprotective lipoprotein into a dysfunctional form, raising the possibility that the enzyme represents a potential therapeutic target for preventing vascular disease in humans (Shao et al., 2006). MPO activity can be measured in blood and tissues by spectrophotometric assays using hydrogen peroxide and o-dianisidine dihydrochloride as substrates. In addition, MPO content can be measured in neutrophils as an index of degranulation with the Coulter counter and flow cytometry and circulating MPO by ELISA. Commercial methods allowing low-cost and high-volume measurements have been proposed. The introduction of these methods of measurement might make MPO a new and useful cardiac biomarker (Valentina et al., 2008). 47 Review of literature Furthermore, high levels of MPO-mediated endothelial dysfunction may be an important mechanistic link between oxidation, inflammation, and cardiovascular disease (CVD) .An elevated level of plasma MPO served as independent predictor of increased risk of myocardial infarction . However, the role of MPO in chronic kidney disease (CKD) is poorly understood, and not much data is available regarding the variations of this enzyme in these patients. It was speculated that raised levels of this enzyme might be one of the factors responsible for the increased risk that these patients have for developing CVD (Madlusudhana, et al., 20l1). Myeloperoxidase (MPO) is a peroxidase enzyme that in humans is encoded by the MPO gene. Myeloperoxidase is most abundantly expressed in neutrophil granulocytes . It is a lysosomal protein stored in azurophilic granules of the neutrophil. MPO has a heme pigment, which causes its green color in secretions rich in neutrophils, such as pus and some forms of mucus )Klebanoff., 2005). Structure: The 150-kDa MPO protein is a dimer consisting of two 15-kDa light chains and two variable-weight glycosylated heavy chains bound to a prosthetic heme group. Three isoforms have been identified, differing only in the size of the heavy chains. It contains a calcium binding site with seven ligands, forming a pentagonal pyramid conformation. One of the ligands is thecarbonyl group of Asp 96. Calcium-binding is important for structure of the active site because of Asp 96's close proximity to the catalytic His95 side chain (Mathy-Hartert et al., 1998). Function: MPO produces hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride anion (Cl-) (or the equivalent from a non48 Review of literature chlorine halide) during the neutrophil's respiratory burst. It requires heme as a cofactor. Furthermore, it oxidizes tyrosine to tyrosyl radical using hydrogen peroxide as an oxidizing agent. Hypochlorous acid and tyrosyl radical are cytotoxic, so they are used by the neutrophil to kill bacteria and other pathogens (Kettle et al., 1997). Myeloperoxidase deficiency is a hereditary deficiency of the enzyme, which predisposes to immune deficiency. Antibodies against MPO have been implicated in various types of vasculitis,most rominently crescentic glomerulonephritisand Churg-Strauss syndrome. They are detected as perinuclear ANCAs (p-ANCAs), as opposed to the cytoplasmic ANCAs (c-ANCAs) against proteinase-3 (PR3), which are strongly associated with Wegener's granulomatosis. Recent studies have reported an association between myeloperoxidase levels and the severity of coronary artery disease. It has been suggested that myeloperoxidase plays a significant role in the development of the atherosclerotic lesion and rendering plaques unstable (Lau and Baldus ; 2006). Medical uses: An initial 2003 study suggested that MPO could serve as a sensitive predictor for myocardial infarction in patients presenting with chest pain. Since then, there have been over 100 published studies documenting the utility of MPO testing. Heslop et al. 2010 reported that elevated MPO levels doubled the risk for cardiovascular mortality over a 13-year period, and measuring both MPO and CRP (C-reactive protein; a general and cardiac-related marker of inflammation) provided added benefit for risk prediction than just measuring CRP alone. (Heslop et al., 2010). 49 Review of literature Upon neutrophil activation and degranulation these enzymes are released into the plasma. MPO has been considered a surrogate marker of neutrophil activation and plasma levels of the enzyme found to be elevated in patients with coronary heart disease (CHD) and acute coronary syndromes (ACS) (Marshall and Catriona, 2011). Immunohistochemical staining for myeloperoxidase used to diagnose acute myeloid leukemia to demonstrate that the leukemic cells were derived from the myeloid lineage. However, the use of myeloperoxidase staining in this setting has been supplanted by the widespread use of flow cytometry. Myeloperoxidase staining is still important in the diagnosis of myeloid sarcoma, contrasting with the negative staining of lymphomas, which can otherwise have a similar appearance (Kagan et al., 2010). Plasma MPO levels have also been investigated in patients presenting with troponin negative ACS and found to be higher than controls suggesting the early activation of neutrophils. MPO has several potentially deletrious effects including the ability to catalyse the production of several reactive oxidant species such as hypochlorus acid which can chlorinate both proteins and lipids. It also consumes nitric oxide affecting endothelial function leading to vasoconstriction (Marshall and Catriona., 2011). In recent times MPO is being implicated in diseases associated with chronic non-microbial pathological processes, which have no direct link with infection, and, in which oxidative stress and inflammation play dominant roles. This article seeks to provide a bird’s eye view of these two aspects of the action of MPO, namely its protective action against 50 Review of literature micro-organisms and its role in chronic diseases associated with inflammation. MPO is an oxidoreductase (EC No. 1.11.1.7) which is stored in the azurophilic granules of polymorphonuclear neutrophils (Usha et al., 2012). The enzyme myeloperoxidase (MPO) was almost exclusively associated with the phenomenon of innate immunity. For almost a century, most of the studies that were carried out on this enzyme were directed towards elucidating the intricate biochemical mechanisms involved in bacterial killing (Iyer et al. 1961). It is a strongly cationic hemoprotein with a molecular mass of 114 kDa. It consists of two identical 72 kDa monomers linked by a disulphide bridge. Each monomer is composed of a light chain and a heavy chain which is glycosylated and also contains the heme lodged in a deep cleft (Usha et al., 2012). Hypochlorous acid further reacts with H2O2 and nitrates to form reactive oxygen and nitrogen species. The highly reactive nature of these products ensures destruction of the invading pathogen and is almost invariably associated with a certain degree of damage to the host tissue. While these reactions are primarily meant to take place within the confines of the phagosomes, not infrequently they are discharged outside the cell and this could bring about destruction of biomolecules in the surrounding tissue. Although MPO is crucial for the protection against invading pathogens, inappropriate activity of this enzyme could lead to host tissue damage. Increased activity of this enzyme is now being implicated in a wide variety of pathological conditions such as 51 Review of literature cardiovascular disease, cancer, renal disease, lung injury and Alzheimer’s disease (Klebenoff , 2005). MPO and Cardiovascular Disease: In recent years several epidemiologic studies have shown that higher levels of MPO are associated with increased risk of cardiovascular disease and also that this was independent of the hitherto established classical risk factors MPO levels were found to be higher in patients angiographically proven coronary artery disease (Usha et al., 2012). MPO in patients presenting with acute chest pain provided clinically useful information of prognostic significance. In patients presenting with acute chest pain, it has been proved that, a single initial measurement of plasma myeloperoxidase independently predicts the early risk of myocardial infarction, as well as the risk of major adverse cardiac events in the ensuing 30-day and six-month periods (Brennan et al., 2003). MPO serves not only as a marker of acute coronary syndromes but is also intimately involved in the process of atherosclerosis; it potentially acts as a mechanistic bridge between inflammation and cardio-vascular disease. MPO dependant processes are involved in the etiopathogenesis of atherosclerosis through multiple mechanisms which include, foam cell formation, endothelial dysfunction, development of vulnerable plaque and ventricular remodeling following acute myocardial infarction (Usha et al .,2012). The oxidative stress created by the down-stream products of increased MPO activity can bring about conversion of native LDL into 52 Review of literature the oxidized LDL, rendering it more atherogenic (Stefanescu et al., 2008). Nitric oxide, a biomolecule which brings about vasodilatation, is rapidly inactivated by products of the MPO reaction causing endothelial dysfunction. MPO also has a role in destabilization of stable coronary plaques by promoting the degradation of the collagen layer which prevents abrupt rupture. Plaque destabilization and rupture are thought to be essential processes in inducing acute cardiovascular events (Usha et al., 2012). MPO and Lung Injury: Studies carried out in experimental animals and humans have shown that MPO has a role to play in the induction of lung injury. Severe acute lung injury has been produced in rats by the simultaneous intratracheal infusion of glucose oxidase which served as a source of H2O2 and MPO Patients with idiopathic pulmonary fibrosis have increased levels of MPO in the alveolar epithelial lining fluid .Tracheal aspirates of premature infants who developed chronic lung disease had elevated levels of 3-chlorotyrosine, which is considered to be a marker of protein damage by the MPO system I (Usha et al., 2012). Filtration of lungs by neutrophils, a condition referred to as neutrophilia, is a common feature in a variety of lung diseases such as acute respiratory distress syndrome, idiopathic pulmonary fibrosis, asbestosis and chronic obstructive pulmonary disease (Haegens, et al 2009). MPO promotes the development of lung neutrophilia and indirectly influences subsequent chemokine and cytokine production in the lung. 53 Review of literature Increased levels of MPO, a marker of active neutrophilia, have been found in the broncho-alveolar lavage of patients with COPD and MPO (Barczyk et al., 2004). MPO and Alzheimer’s Disease: MPO has been detected in microglia adjacent to senile clots in the cerebral cortex of patients with Alzheimer’s disease Apo-E which is also found in senile clots of these patients is highly susceptible to oxidation by MPO (Reynolds et al., 1999). MPO and Kidney Disease MPO has been shown to be an important pathogenic factor in glomerular and tubulo-interstitial diseases. Several studies have shown the presence of MPO-containing cells MPO activity triggers the production of several highly reactive and deleterious products as well as MPO activity in a variety of renal disorders (Usha et al .,2012). When neutrophils adhere to glomeruli, they generate oxidants through MPO-catalysed reactions, causing degradation of the glomerular basement membrane. Renal perfusion experiments with MPO followed by its substrates, namely, H2O2 and chloride ions, resulted in glomerular morphologic changes, endothelial and mesangial cell injury, activation of platelets, and subsequent proliferative responses mimicking inflammatory and proliferative glomerular nephritis in humans. MPO has been implicated in the pathogenesis of various types of renal diseases (Malle et al., 2003). 54 Review of literature Lipoprotien (a) Lipoprotein(a) [Lp(a)] has been considered a cardiovascular risk factor for many years. Owing to incomplete scientific evidence, screening for and treatment of high Lp(a) levels have to date been performed principally by lipid specialists. However, during the last few years, major advances have been achieved in understanding the causal role of elevated Lp(a) in premature cardiovascular disease (CVD) (Kamstrup et al., 2009). Lipoprotein (a) is a plasma lipoprotein consisting of a cholesterolrich LDL particle with one molecule of apolipoprotein B100 and an additional protein, apolipoprotein (a), attached via a disulfide bond. Elevated Lp(a) levels can potentially increase the risk of CVD (i) via prothrombotic/anti-fibrinolytic effects as apolipoprotein(a) possesses structural homology with plasminogen and plasmin but has no fibrinolytic activity and (ii) via accelerated atherogenesis as a result of intimal deposition of Lp(a) cholesterol, or both (Clarke et al., 2009). Genetics: Plasma levels of Lp(a) are to a large extent genetically determined via variation in the apolipoprotein(a) gene This makes the apolipoprotein(a) gene ideal for use in a Mendelian randomization study, examining whether lifelong, genetically elevated levels of plasma Lp(a) cause CVD. By analogy, familial hypercholesterolaemia with mutations in the LDL receptor or apolipoprotein B genes have lifelong, genetically elevated LDL cholesterol levels and premature CVD, a fact that has helped establish that elevated LDL cholesterol levels constitute a direct cause of atherosclerosis and CVD (Borge et al., 2010). 55 Review of literature A Mendelian randomization study needs three pieces of data to help provide evidence for a causal link between elevated plasma Lp(a) levels and CVD. First, elevated plasma Lp(a) levels is associated with increased CVD risk. Secondly, genetic variation should exist in human populations that can explain a large fraction of the variation in plasma Lp(a) levels: such genetic variation has been known for many years, most importantly the kringle IV type 2 size polymorphism ,resulting in a variable number from 2 to >40 number of a 5.6 kb repeat associated inversely with plasma Lp(a) levels. Thus, the fewer the repeats in the apolipoprotein(a) gene, the higher the plasma levels of Lp(a), which has also been demonstrated in the past. Thirdly, such genetic variation should be linked directly with CVD risk (Kamstrup et al., 2009). Metabolism: It is believed that plasma concentrations of Lp(a) are determined chiefly by rates of hepatic synthesis of apolipoprotein(a): although the site of formation of Lp(a) has not been definitively identified, evidence suggests that apolipoprotein(a) adducts extracellularly and covalently to apolipoprotein B100-containing lipoproteins, predominantly LDL, Apolipoprotein(a) genotype, which determines both the synthetic rate and size of the apolipoprotein(a) moiety of Lp(a), alone accounts for 90% of plasma concentrations of Lp(a) (Borge et al., 2010). As hepatic secretion rates are lower for large apolipoprotein(a) isoforms, and as most individuals are heterozygous for two different isoforms, the smallest isoform typically predominates in plasma. Lipoprotein(a) is thought to be catabolized primarily by hepatic and renal pathways, but these metabolic routes do not appear to govern plasma Lp(a) levels (Ballantyne et al., 2009). 56 Review of literature Pathophysiological Mechanisms Underlying the Atherothrombotic Potential of Lipoprotein (a): After transfer from plasma into the arterial intima, Lp(a) may be more avidly retained than LDL as it binds to the extracellular matrix not only through apolipoprotein(a), but also via its apolipoprotein B component, thereby contributing cholesterol to the expanding atherosclerotic plaque. In vitro, Lp(a) binds to several extracellular matrix proteins including fibrin and defensins, a family of 29–35 amino acid peptides that are released by neutrophils during inflammation and severe infection. It is likely that defensins, like lipoprotein lipase, provide a bridge between Lp(a) and the extracellular matrix (Nielsen, 1999). Lp(a) also interacts with the β2-integrin Mac-1, thereby promoting the adhesion of monocytes and their transendothelial migration. [25] In atherosclerotic coronary arteries, Lp(a) was found to localize in close proximity to Mac-1 on infiltrating mononuclear cells (Sotiriou et al., 2006). Lipoprotein(a) has also been shown to bind pro-inflammatoryoxidized phospholipids and is a preferential carrier of oxidized phospholipids in human plasma. Lipoprotein(a) also contains lipoproteinassociated phospholipase A2 (equally referred to as Paf-acetylhydrolase), which may cleave oxidized fatty acids at the sn-2 position in oxidized phospholipids to yield short chain fatty acids and lysolecithin (Tsimikas et al., 2007).] Apolipoprotein(a), a homologue of the fibrinolytic proenzyme plasminogen, impairs fibrinolysis. Indeed, Lp(a)/apolipoprotein(a) can competitively inhibit tissue-type plasminogen activator-mediated plasminogen activation on fibrin surfaces, although the mechanism of inhibition by apolipoprotein(a) remains controversial. Essential to fibrin 57 Review of literature clot lysis are a number of plasmin-dependent, positive feedback reactions that enhance the efficiency of plasminogen activation, including the plasmin-mediated conversion of Glu-plasminogen to Lys-plasminogen. It has been observed that the apolipoprotein(a) component of Lp(a) inhibits the key positive feedback step involving conversion of plasmin-mediated Glu-plasminogen to Lys-plasminogen. Lipoprotein(a) may also enhance coagulation by inhibiting the function of tissue factor pathway inhibitor (Feric et al., 2008). Small isoforms of apolipoprotein(a) have been observed to possess elevated potency in inhibiting fibrinolysis and thereby promoting thrombosis. Indeed, a recent meta-analysis demonstrated a two-fold increase in the risk of CHD and ischaemic stroke in subjects with small apolipoprotein(a) phenotypes. Furthermore, prospective findings in the Bruneck study have revealed a significant association specifically between small apolipoprotein(a) phenotypes and advanced atherosclerotic disease involving a component of plaque thrombosis. These data suggest that the determination of apolipoprotein(a) phenotype/genotype may provide clinicians with additional information by which to evaluate Lp(a)/apolipoprotein(a)-associated atherothrombotic risk (Børge et al., 2010). Elevated Lp(a) levels may promote atherosclerosis via Lp(a)derived cholesterol entrapment in the intima, via inflammatory cell recruitment, and/or via the binding of pro-inflammatory-oxidized phospholipids. The prothrombotic, anti-fibrinolytic actions of apolipoprotein(a) are expressed on the one hand as inhibition of fibrinolysis with enhancement of clot stabilization and on the other as enhanced coagulation via the inhibition of tissue factor pathway inhibitor (Erqou et al., 2010). 58 Review of literature Measurement: Several types of Lp(a) assays are currently available, some commercially; prominent among them are sandwich enzyme-linked immunosorbent assays (ELISA), non-competitive ELISA, latex immunoassays, immunonephelometric assays, and immunoturbidometric and fluorescence assays. In order for clinical laboratories to provide clinicians with Lp(a) values which allow correct cardiovascular risk evaluation when Lp(a) is included in the estimate, the following elements in standardization between Lp(a) assays are critical (Børge et al., 2010). Compared with LDL, Lp(a) is relatively refractory to both lifestyle and drug intervention. The data on the effects of statins and fibrates on Lp(a) are limited and highly variable. Overall, statins have, however, been shown to consistently and modestly decrease elevated Lp(a) in patients with heterozygous familial hypercholesterolaemia. Other agents reported to decrease Lp(a) to a minor degree (<10%) include aspirin, Lcarnitine, ascorbic acid combined with L-lysine, calcium antagonists, angiotensin-converting enzyme inhibitors, androgens, oestrogen, and its replacements (e.g. tibolone), anti-estrogens (e.g. tamoxifen), and thyroxine replacement in hypothyroid subjects (Ballantyne ,et al., 2009). In the absence of a defined physiological role of Lp(a), its pathophysiological role is undoubtedly that of a prominent risk factor for the development of CVD. Circulating levels of Lp(a) are not significantly modified by traditional lipid-lowering therapies, and so alternative approaches to target its adverse functions specifically are necessary and may be of therapeutic value. This paper will focus on the detrimental effects of Lp(a) in the cardiovascular system including the coagulation cascade, inflammatory pathways and modulation of smooth muscle 59 Review of literature (SMC), and endothelial cell (EC) behaviour within blood vessel walls (Lippi and Targher, 2012). Following injury to the vessel wall, platelets become activated and trigger thrombus formation. Fibrin cross-links and stabilises the clot; during resolution it is broken down by plasmin to minimise vessel occlusion. Lp(a) has been demonstrated to act as a prothrombotic factor, interfering with clot biology at multiple levels, as follows: Evidence of Lp(a) influencing the initial activation of platelets is scarce, although both Lp(a) and apo(a) alone have been demonstrated to promote activation via thrombin-receptor-activated hexapeptide (TRAP). However, the ability of Lp(a) to directly affect platelet aggregation is much more contentious. Studies have shown that both Lp(a) and apo(a) alone enhanced aggregation in response to arachidonic acid and TRAP had no effect on aggregation induced by collagen or thrombin (Riches and Porter,2012). Lp(a) had previously been demonstrated to inhibit aggregation induced by low concentrations of collagen (4 mg/mL) ,however, in that case the inhibitory effect was not observed when collagen concentrations were increased to 10 mg/mL . Aggregation in response to platelet activating factor (PAF) has also been reported to be inhibited by Lp(a) ( Tsironis et al., 2004). The antiaggregatory effects of Lp(a) may be mediated via its interaction with integrin I I b3. Integrin I I b3 is normally bound by fibrinogen to promote platelet aggregation, yet apo(a) can displace fibrinogen from the receptor thus inhibiting this process. In addition, 60 Review of literature functional effects of Lp(a) can be dependent on modifications of the Lp(a) molecule—platelet granule secretion was altered when Lp(a) was modified by lipid peroxidation products or acetylation . It is clear that the interaction of Lp(a) with platelets is complex and involves a balance between Lp(a) subunit binding, protein modifications, and the factor stimulating platelet aggregation (Riches and Porter,2012) Transportation of Oxidised Phospholipids: Lp(a) is claimed to be an acute phase reactant, with increased circulating levels being observed following myocardial infarction and percutaneous coronary intervention. It is speculated that this may point to a physiological anti-inflammatory role for Lp(a) in patients with low plasma levels, whereby Lp(a) could bind to and remove oxidised phospholipids from the circulation, preventing further damage. Oxidised phospholipids are proinflammatory in nature and are bound by Lp(a). Although they are often found associated with apoB-100 , studies have shown that within the Lp(a) molecule the association was dependent on KV of the apo(a) moiety (Edelstein et al., 2003). The amount of oxidised phospholipid bound to apo(a) remained constant and was unaffected by apo(a) size suggesting that it was bound to apo(a) during synthesis in the hepatocyte and was not derived from plasma LDL. Whilst this may be beneficial in low concentrations, in patients with high plasma levels of Lp(a) preferential binding of oxidised phospholipids may lead to their deposition within the vessel wall, hence promoting atherogenesis (Edelstein et al., 2009). 61 Review of literature Induction of Inflammatory Cytokines Lipoprotein(a) (Lp(a)) is an LDL-like molecule consisting of an apolipoprotein B-100 (apo(B-100)) particle attached by a disulphide bridge to apo(a). Many observations have pointed out that Lp(a) levels may be a risk factor for cardiovascular diseases. Lp(a) inhibits the activation of transforming growth factor (TGF) and contributes to the growth of arterial atherosclerotic lesions by promoting the proliferation of vascular smooth muscle cells and the migration of smooth muscle cells to endothelial cells (Malaguarnera et al, 2013). Lp(a) has been shown to induce inflammatory cytokine expression in a cell-type-specific manner. For example, apo(a) induced IL-8 expression in macrophages, but not monocytes. Detailed analysis revealed that Lp(a) induced a 12-fold increase in IL-8 mRNA, whereas apo(a) alone was almost three times more potent in inducing transcription. This was mirrored at the protein level and was dependent on KV and interaction with Gs protein receptors. IL-8 induction was not observed by exposure to LDL or Lp(a) moieties without the apo(a) fragment confirming the essential role of the apo(a) moiety in this process. In addition, Lp(a) also induced expression of IL-1β, tumour necrosis factor alpha (TNF-α), and monocyte chemoattractant protein (MCP-1) in macrophages (Nakagami et al., 2010). Moreover Lp(a) inhibits plasminogen binding to the surfaces of endothelial cells and decreases the activity of fibrin-dependent tissue-type plasminogen activator. Lp(a) may act as a proinflammatory mediator that augments the lesion formation in atherosclerotic plaques. Elevated serum Lp(a) is an independent predictor of coronary artery disease and myocardial infarction (Malaguarnera et al, 2013). 62 Review of literature Cardiovascular diseases cause 3% of all deaths in North America being the most common cause of death in European men under 65 years of age and the second most common cause in women. These facts suggested us to consider new strategies for prediction, prevention, and treatment of cardiovascular disease (Klingenberg and Hansson et al., 2009). Furthermore, Lp(a) levels should be a marker of restenosis after percutaneous transluminal coronary angioplasty, saphenous vein bypass graft atherosclerosis, and accelerated coronary atherosclerosis of cardiac transplantation. Finally, the possibility that Lp(a) may be a risk factor for ischemic stroke has been assessed in several studies. Recent findings suggest that Lp(a)-lowering therapy might be beneficial in patients with high Lp(a) levels. A future therapeutic approach could include apheresis in high-risk patients in order to reduce major coronary events (Malaguarnera et al, 2013). Inflammatory mechanisms play a central role in the pathogenesis of atherosclerosis and its complications .It has been demonstrated that atherogenic lipoproteins such as apo(B-100), oxidized low-density lipoprotein (LDL), remnant lipoprotein (beta-VLDL), and lipoprotein(a) play a critical role in the proinflammatory reaction. High-density lipoprotein (HDL) is antiatherogenic lipoproteins that exert antiinflammatory functions (Motta et al., 2009). Plasma LDL cholesterol is a well-established predictor of coronary artery disease (CAD), and many observations have pointed out that Lp(a) and apolipoprotein(a) (apo(a)) levels may be risk factors for cardiovascular diseases (CVD) (Walldius and Jungner, 2004). 63 Review of literature Animal experiments showed that apo(a) serves as a distinctive marker of Lp(a) and represents an atherogenic component of Lp(a) .Furthermore, apo(a) has also been reported to be correlated to coronary artery disease as well as renal disease. Dissociation of apo(a) may lead to the exposure of an additional lysine-binding site, increasing the affinity of free apo(a) for plasmin modified fibrin, thus impeding fibrinolysis .Apo(a) is a member of a family of “kringle" containing proteins, such as plasminogen, tissue plasminogen activator (tPA), prothrombin, factor XII, and macrophage stimulating factor (MSF). Lp(a) shares a high degree of sequence identity with plasminogen. These similarities could explain the role of Lp(a) in thrombogenesis and as a proinflammatory factor .Native Lp(a) has been shown to enhance the expression of adhesion molecules (Galvano et al., 2010). Because of the structural homology with plasminogen, Lp(a) might have important antithrombolytic properties, which could contribute to the pathogenesis of atherothrombotic disease. For example, Lp(a) binding to immobilised fibrinogen and fibrin results in the inhibition of plasminogen binding to these substrates .In addition, Lp(a) competes with plasminogen for its receptors on endothelial cells, leading to diminished plasmin formation, thereby delaying clot lysis and favouring thrombosis. The high affinity of Lp(a) for fibrin provides a mechanistic basis for their frequent colocalization in atherosclerotic plaques .Moreover Lp(a) induces the monocyte chemoattractant (CC chemokine I-309), which leads to the recruitment of mononuclear phagocytes to the vascular wall (Haque et al., 2000). 64 Review of literature Oxidized Lp(a): Lp(a) particles can suffer oxidative modification and scavenger receptor uptake, with cholesterol accumulation and foam cell formation ,leading to atherogenesis. Oxidation of LDL and Lp(a) affects the catabolism of the lipoproteins, including changes in receptor recognition, catabolic rate, retention in the vessel wall, and propensity to accelerate atherosclerosis. Oxidative modification of apo(a) may have an influence on Lp(a) recognition by scavenger receptors of macrophages. Some studies showed that Lp(a) particles are prone to oxidation and that the increased risk of coronary artery disease associated with elevated Lp(a) levels may be related in part to their oxidative modification and uptake by macrophages, resulting in the formation of macrophage-derived foam cells (Malaguarnera et al, 2013). The oxidative form of Lp(a) (ox-Lp(a)) might attenuate fibrinolytic activity through the reduction of plasminogen activation, might enhance PAI-1 production in vascular endothelial cells, and might impair endothelium-dependent vasodilation. Particularly, the role of ox-Lp(a) is linked to macrophages that take up ox-Lp(a) via scavenger receptor as well as oxidized LDL. Lp(a) particles are susceptible to oxidative modification and scavenger receptor uptake, leading to intracellular cholesterol accumulation and foam cell formation, which contributes further to atherogenesis .Morishita et al. demonstrated increased values of ox-Lp(a) in patients with coronary artery disease (Morishita et al., 2009). A study of autopsy findings demonstrated a deposition of ox-Lp(a) in the vessel margin inside the calcified areas .Probably it was related to the promotion of an antifibrinolytic environment, foam cell formation, generation of a fatty streak, and an increase in smooth muscle cells. 65 Review of literature Moreover ox-Lp(a) is a potent stimulus of monocyte adhesion to endothelial cells, thus contributing to atherogenic changes in human blood vessels. (Malaguarnera et al, 2013). Komai et al., 2002 compared the effects of oxidized lipoproteins and no oxidized lipoprotein on the progression of atherosclerosis. It was investigated the mitogenic actions of Lp(a) and ox-Lp(a) on human vascular smooth muscle cells (VSMC). The results were that Lp(a) significantly stimulated the growth of human VSMC in a dose-dependent manner, whereas ox-Lp(a) showed a stronger stimulatory action on VSMC growth than native Lp(a). This study demonstrated that ox-Lp(a) has a more potent effect than native Lp(a) in developing atherosclerosis diseases. Glycated Lp(a): Nonenzymatic glycation of lipoprotein may contribute to the premature atherogenesis in patients with diabetes mellitus by diverting lipoprotein catabolism from nonatherogenic to atherogenic pathways. It has been observed that the proportion of apo (B-100) in glycated form was significantly higher in diabetic patients than in nondiabetic controls, and equally that plasma Lp(a) levels might be increased in diabetic patients .Anyway, glycation does not appear to significantly enhance the atherogenic potential of unmodified Lp(a) ( Libby, 2002). The formation of advanced glycation end-products (AGEs) and EC-mediated oxidative modification may contribute to the alterations of the generation of PAI-1 and t-PA induced by glycated Lp(a) . The combination of hyperglyaemia and hyperlipoprotein(a) may reduce ECderived fibrinolytic activity, which may promote the development of 66 Review of literature thrombosis and atherosclerosis in subjects with diabetes (Malaguarnera et al., 2013). A task force for emerging risk factor assessed the relationship between Lp(a) concentration and risk of major vascular and nonvascular outcomes. In this long-term prospective study, Lp(a) plasma levels and subsequent major vascular morbidity and/or cause-specific mortality were recorded. Lp(a) was weakly correlated with several conventional vascular risk factors, and it was highly consistent within individuals over several years (Erqou et al., 2009). Several epidemiologic studies have assessed the association between Lp(a) and atherosclerotic disease .Many population-based prospective studies had reported a controversial association between Lp(a) levels and CHD risk. Few studies, however, have adequately examined important aspects of the association, such as the size of relative risks in clinically relevant subgroups (such as in men and women or at different levels of established risk factors) (Malaguarnera et al., 2013). 67 Review of literature Highly sensitive c-reactive protien C-reactive protein (CRP) is a protein found in the blood, the levels of which rise in response to inflammation (i.e. C-reactive protein is an acute-phase protein). Its physiological role is to bind to phosphocholine expressed on the surface of dead or dying cells (and some types of bacteria) in order to activate the complement system . CRP is synthesized by by macrophages and the liver in fat cells response (adipocytes). It to factors is a released member of the pentraxin family of proteins. It is not related to C-peptide or protein C. C-reactive protein was the first pattern recognition receptor (PRR) to be identified. (Mantovani et al., 2008). History and nomenclature C- reactive protein (CRP) was so named because it was first discovered as a substance in the serum of patients with acute inflammation that reacted with the C- (capsular ( polysaccharide of pneumococcus ,Discovered by Tillett and Francis in 1930, it was initially thought that CRP might be a pathogenic secretion as it was elevated in people with a variety of illnesses including cancer ,however, the discovery of hepatic synthesis demonstrated that it is a native protein . (Peter et al., 2009). Genetics and biochemistry: The CRP gene is located on the first chromosome (1q21-q23). CRP is a 224-residue protein with a monomer molecular mass of 25106 Da. The protein is an annular pentameric disc in shape and a member of the small pentraxins family (Kennelly et al., 2009). 68 Review of literature Function: (Pepys and Hirschfield, 2003). The acute phase response develops in a wide range of acute and chronic inflammatory conditions like bacterial, viral, or fungal infections; rheumatic and other inflammatory diseases; malignancy; and tissue injury or necrosis. These conditions cause release of interleukin-6 and other cytokines that trigger the synthesis of CRP and fibrinogen by the liver. During the acute phase response, levels of CRP rapidly increase within 2 hours of acute insult, reaching a peak at 48 hours. With resolution of the acute phase response, CRP declines with a relatively short half-life of 18 hours. Measuring CRP level is a screen for infectious and inflammatory diseases. Rapid, marked increases in CRP occur with inflammation, infection, trauma and tissue necrosis, malignancies, and autoimmune disorders. Because there are a large number of disparate conditions that can increase CRP production, an elevated CRP level does not diagnose a specific disease. An elevated CRP level can provide support for the presence of an inflammatory disease, such as rheumatoid arthritis, polymyalgia rheumatica or giant-cell arteritis. CRP binds to phosphocholine on microbes. It is thought to assist in complementbinding to foreign and damaged cells and enhances phagocytosis by macrophages (opsonin mediated phagocytosis), which express a receptor for CRP. It is also believed to play another important role in innate immunity, as an early defense system against infections. CRP rises up to 50,000-fold in acute inflammation, such as infection. It rises above normal limits within 6 hours, and peaks at 48 hours. Its half-life is constant, and therefore its level is mainly determined by the rate of production (and hence the severity of the precipitating 69 Review of literature cause). Serum amyloid A is a related acute-phase marker that responds rapidly in similar circumstances. The physiological role of CRP is to bind to phosphocholine expressed on the surface of dead or dying cells (and some types of bacteria) in order to activate the complement system. CRP binds to phosphocholine on microbes and damaged cells and enhances phagocytosis by macrophages. Thus, CRP participates in the clearance of necrotic and apoptotic cells. CRP is a member of the class of acute-phase reactants, as its levels rise dramatically during inflammatory processes occurring in the body. This increment is due to a rise in the plasma concentration of IL-6, which is produced predominantly bymacrophages as well as adipocytes (Lau et al., 2005). Clinical significance: Scleroderma, polymyositis, and dermatomyositis often elicit little or no CRP response. CRP levels also tend not to be elevated in SLE unless serositis or synovitis is present. Elevations of CRP in the absence of clinically significant inflammation can occur in renal failure. CRP level is an independent risk factor for atherosclerotic disease. Patients with high CRP concentrations are more likely to develop stroke, myocardial infarction, and severe peripheral vascular disease (LopezGarcia et al., 2005). Role in cardiovascular disease: Research suggests that patients with elevated basal levels of CRP are at an increased risk of diabetes, hypertension and cardiovascular disease. A study of over 700 nurses showed that those in the highest quartile of trans fat consumption had blood levels of CRP that 70 Review of literature were 73% higher than those in the lowest quartile (Lopez-Garcia et al., 2005). Although one group of researchers indicated that CRP may be only a moderate risk factor for cardiovascular disease, this study (known as the Reykjavik Study) was found to have some problems for this type of analysis related to the characteristics of the population studied, and there was an extremely long follow-up time, which may have attenuated the association between CRP and future outcomes. Others have shown that CRP can exacerbate ischemic necrosis in a complement-dependent fashion and that CRP inhibition can be a safe and effective therapy for myocardial and cerebral infarcts; so far, this has been demonstrated in animal models only (Pepys et al., 2006). It has been hypothesized that a high CRP levels might reflect a large benefit from statins. This is based on the JUPITER trial that found that elevated CRP levels without hyperlipidemia benefited. Statins were selected because they have been proven to reduce levels of CRP . Studies comparing effect of various statins in hs-CRP revealed similar effects of different statins. A subsequent trial however failed to find that CRP was useful for determining statin benefit (Emberson et al., 2011). Role in cancer: The role of inflammation in cancer is not well understood. Some organs of the body show greater risk of cancer when they are chronically inflamed. Blood samples of persons with colon cancer have an average CRP concentration of 2.69 milligrams per liter. Persons without colon cancer average 1.97 milligrams per liter. The difference was statistically significant. These findings concur with previous studies that indicate that anti-inflammatory drugs could lower colon cancer risk (Erlinger et al., 2004). 71 Review of literature Diagnostic use: Measuring and charting CRP values can prove useful in determining disease progress or the effectiveness of treatments. Normal concentration in healthy human serum is usually lower than 10 mg/L, slightly increasing late pregnant women, with aging. Higher levels mild inflammation and viral are found in infections(10– 40 mg/L), active inflammation, bacterial infection (40–200 mg/L), severe bacterial infectionsand burns (>200 mg/L) (Clyne and Olshaker, 1999). CRP is used mainly as a marker of inflammation. Apart from liver failure, there are few known factors that interfere with CRP production. Various analytic methods are available for CRP determination, such as enzyme-linked immunosorbent assay (ELISA), immunoturbidimetry, rapid immunodiffusion, and visual agglutination. In general, both the CRP test and another test, called the erythrocyte sedimentation rate (ESR), measure the increase in inflammatory generated proteins. The CRP test is a direct measurement of C-reactive protein, while ESR indirectly measures many proteins associated with inflammation (Saljoughian, 2008) CRP is a more sensitive and accurate reflection of the acute phase response than the ESR (Erythrocyte Sedimentation Rate). The half-life of CRP is constant. Therefore, CRP level is mainly determined by the rate of production (and hence the severity of the precipitating cause). In the first 24 h, ESR may be normal and CRP elevated. CRP returns to normal more quickly than ESR in response to therapy (Clyne and Olshaker, 1999). 72 Review of literature Cardiology diagnostic test: Arterial damage results from white blood cell invasion and inflammation within the wall. CRP is a general marker for inflammation and infection, so it can be used as a very rough proxy for heart disease risk. Since many things can cause elevated CRP, this is not a very specific prognostic indicator (Goldman, 2011). The American Heart Association and U.S. Centers for Disease Control and Prevention have defined risk groups as follows: - Low Risk: less than 1.0 mg/L. - Average risk: 1.0 to 3.0 mg/L. - High risk: above 3.0 mg/L But hs-CRP is not to be used alone and should be combined with elevated levels of cholesterol, LDL-C, triglycerides, and glucose level. Smoking, hypertension and diabetes also increase the risk level of cardiovascular disease (Swardfager et al., 2012). Nevertheless, a level above 2.4 mg/L has been associated with a doubled risk of a coronary event compared to levels below 1 mg/L; however, the study group in this case consisted of patients who had been diagnosed with unstable angina pectoris; whether elevated CRP has any predictive value of acute coronary events in the general population of all age ranges remains unclear. Currently, C-reactive protein is not recommended as a cardiovascular disease screening test for average-risk adults without symptoms (Goldman, 2011). The level of this protein in plasma increases greatly during acute phase response to tissue injury ,infection, or other inflammatory stimuli. It is induced by interleukin-1 and interleukin-6 (Faraj and Salem, 2012). 73 Review of literature There are two different tests for CRP. The standard test measures a much wider range of CRP levels but is less sensitive in the lower ranges. The high-sensitivity CRP (hs-CRP) test can more accurately detect lower concentrations of the protein (it is more sensitive), which makes it more useful than the CRP test in predicting a healthy person's risk for cardiovascular disease (Clyne and Olshaker, 1999). CRP is one of several proteins that are often referred to as acute phase reactants and is used to monitor changes in inflammation associated with many infectious and autoimmune diseases (Faraj and Salem, 2012). C-reactive protein (CRP) is a substance that is released into the blood in response to inflammation, the process by which the body responds to injury. Elevated levels of CRP in the blood mean that there is inflammation somewhere in the body, but other tests are needed to determine the cause and location of the inflammation. Physicians now believe that atherosclerosis, or hardening of the arteries, is an inflammatory process. Atherosclerosis causes only a small amount of CRP to be released into the blood. Therefore, a very sensitive test called a high-sensitivity CRP test (hs-CRP) is used to measure CRP levels (Ridker et al., 2007). 74