Chapter one: Introduction ……………………........................................................................1
1.1 Atherosclerosis:
Atherosclerosis is a disease of large and medium-sized muscular arteries and it
is characterized by endothelial dysfunction, vascular inflammation, and the
buildup of lipids, cholesterol, calcium, and cellular debris within the intima of
the vessel wall. This buildup results in plaque formation, vascular remodeling,
acute and chronic luminal obstruction, abnormalities of blood flow, and
diminished oxygen supply to target organs(1,2).
1.1.2. Mortality/Morbidity:
Atherosclerosis is predicted to be the leading cause of death in the developing
world within the first quarter of the next century. Also arteriosclerosis of the
coronary and peripheral vasculature is the leading cause of death among men
and women in the United States and worldwide(3,4).Estimates for 2005 in the
United States alone are that about 16 million people have atherosclerotic heart
disease and 5.8 million have stroke. Cardiovascular disease, primarily coronary
and cerebrovascular atherosclerosis, caused almost 870,000 deaths in 2005almost twice as many as cancer caused and 9 times as many as injuries
caused(5).
1.1.3 Atherosclerosis risk factors(6,7,8):
There are many risk factors of atherosclerosis which either:
1. Non-modifiable like Age, gender and genetic.
2. Modifiable like dys-lipidaemia, hypertension, diabetes, infection, smoking,
hyperuriceamia , drugs, obesity and systemic autoimmune disease.
Chapter one: Introduction ……………………........................................................................2
Although the risk factors for atherosclerosis are different; they are identical in
the presence of inflammatory state that is responsible for atherosclerotic
complication.
1.2 Pathogenesis of atherosclerosis:
Atherosclerosis describing the association of fatty degeneration and vessel
stiffening. This process is characterized by patchy intramural thickening of the
subintima that encroaches on the arterial lumen.(9)
The proposed initial step in atherogenesis is endothelial dysfunction leading to
a number of compensatory responses that alter the normal vascular homeostatic
properties(10).
The term endothelial dysfunction refers to an imbalance in the production or
bioavailability of endothelial vasodilator mediators (NO) which lead to altering
the reactivity of the vessel and causing paradoxical vasoconstriction , loss of
the natural antithrombotic properties and of the selective permeability of the
endothelium(11,12). Pro-inflammatory stimuli, including a diet high in saturated
fat,
hypercholesterolemia,
hypertension, and smoking
obesity,
hyperglycemia,
insulin
resistance,
trigger the endothelial expression of adhesion
molecules such as P-selectin, E-selectin, ICAM-1 and
VCAM-1 which
mediate the attachment of circulating monocytes and lymphocyte(13,14).
Atherosclerotic lesions develop as a result of inflammatory stimuli, subsequent
release of various cytokines, proliferation of smooth muscle cells, synthesis of
connective tissue matrix, and accumulation of macrophage and lipid.
Atherosclerosis is likely initiated when endothelial cells over-express adhesion
molecules in response to turbulent flow in the setting of an unfavorable serum
Chapter one: Introduction ……………………........................................................................3
lipid profile. Animals fed a pro-atherogenic diet rapidly over express vascular
cell adhesion molecule-1 (VCAM-1)(15).
Expression of VCAM-1 on endothelial surfaces was an early, and necessary,
step in the pathogenesis of atherosclerosis. Increased cellular adhesion and
associated endothelial dysfunction then "sets the stage" for the recruitment of
inflammatory cells, release of cytokines and recruitment of lipid into the
atherosclerotic plaque(16).
VCAM-1 expression increases recruitment of monocytes and T-cells to sites of
endothelial injury; subsequent release of monocyte chemo-attractant protein-1
(MCP-1) by leukocytes magnifies the inflammatory cascade by recruiting
additional leukocytes, activating leukocytes in the media, and causing
recruitment and proliferation of smooth muscle cells (16) .
However in response to signals generated within the early plaque, monocytes
adhere to the endothelium and then migrate through the endothelium and
basement membrane by elaborating enzymes, including locally activated matrix
metalloproteinase (MMP) that degrade the connective tissue matrix . Recruited
macrophages both release additional cytokines and begin to migrate through the
endothelial surface into media of the vessel. This process is further enhanced
by the local release of monocytes-colony stimulating factor (M-CSF), which
causes monocytic proliferation; local activation of monocytes leads to both
cytokine-mediated progression of atherosclerosis, and oxidation of low-density
lipoprotein (LDL)(17).
Chapter one: Introduction ……………………........................................................................4
1.2.1 Inflammatory mediators in atherosclerosis:
Many mediators of inflammation have been described to influence the
development of the atherosclerotic plaque. For example, CD40L elaborated
within the plaque has been shown to increase the expression of tissue factor in
atherosclerotic plaques.(18)
Inflammatory mediators expressed by smooth cells within the atherosclerotic
plaque include, but are not limited to, interleukin (IL)-1ß, tumor necrosis factor
(TNF) , IL-6, M-CSF, MCP-1, IL-18,IL-17A and CD-40L. The impact of these
mediators
is
diverse
and
includes mitogenesis,
intracellular
matrix
proliferation, angiogenesis and foam cell development . Repeated cycles of
inflammation lead to accumulation of macrophages, some of which can die in
this location, produce so-called necrotic core, and induce smooth muscle cell
(SMC) proliferation and migration in lesion to form fibrous cap of advanced
complicated stable atherosclerotic lesion (stable plaque) (19).
Atherosclerotic lesions are composed of three major components(20).
1. The cellular component comprised predominately of smooth muscle cells
and macrophages.
2. The connective tissue matrix and extracellular lipid.
3. Intracellular lipid that accumulates within macrophages, thereby converting
them into foam cells.
The earliest visible lesion of atherosclerosis is the fatty streak, which is due to
an accumulation of lipid-laden foam cells in the intimal layer of the artery.
Chapter one: Introduction ……………………........................................................................5
With time, the fatty streak evolves into a fibrous plaque, the hallmark of
established atherosclerosis.
Ultimately the lesion may evolve to contain large amounts of lipid; if it
becomes unstable, denudation of overlying endothelium, or plaque rupture,
may result in thrombotic occlusion of the overlying artery.
Macrophages
incorporate lipids from oxidized low density lipoprotein (LDL) via the
scavenger receptor pathway (CD36, CD40) which is tightly regulated by
cytokines IFN-y, TNF-α, and IL-6. After that macrophage becomes foam cells
i.e., lipid-laden macrophages, the hallmark of the early fatty streak lesion (21,22).
The lipid-laden macrophages (foam cells), forming the fatty streak, secrete pro
inflammatory cytokines that amplify the local inflammatory response in the
lesion, matrix metalloproteinase (MMPs), tissue factor into the local matrix,
and growth factors that stimulate the smooth muscle replication responsible for
lesion growth(17).
Localization and accumulation of lipid occurs in response to earlier changes in
the vascular endothelium. Accumulation of lipid is, however, required for the
development of the definitive plaque. Lipid deposition likely starts with the
movement of LDL from the blood into the vessel wall. It may move back into
the bloodstream (a hallmark of lesion regression and a process that may be
facilitated by some lipid lowering strategies), it may become oxidized (through
action of free radicals or direct activity of leukocytes) or it may be taken up by
monocytes/macrophages which ultimately become foam cells. Oxidized LDL is
particularly atherogenic and is chemo tactic for monocytes-macrophages.(23,24).
Chapter one: Introduction ……………………........................................................................6
1.2.2Fate of atherosclerotic plaque:
Evolution of the atherosclerotic plaque is characterized by gradual enlargement
over time due to the accumulation of foam cells. The gradually enlarging
plaque may precipitate chronic stable angina. However, myocardial infarction
oftentimes occurs within vessels with relatively unremarkable narrowing. This
suggests that rapidly growing plaques cause many myocardial infarctions.(25)
Slowly growing plaques gradually accumulate lipid within foam cells;
proliferation of smooth muscle cells and elaboration of intracellular matrix
produce the definitive fibrous plaque. In general, such plaques tend to have
adherent endothelial layers that are not prone to sudden disruption with
associated activation of coagulation. Some plaques grow at a much greater rate
than would be predicted by simple lipid accumulation and expansion of the
components of the fibrous plaque. Cholesterol accumulation within such
plaques is due to both "passive" transfer of LDL from the circulation and
scavenging of red blood cell membranes deposited during intraplaque
hemorrhage.(26)
Two forms of plaque injury are recognized: superficial and deep.
1.Superficial injury produces areas of focal endothelial denudation that can
enlarge and lead to the formation of mural or even occlusive thrombi. Plaques
that are capped with superficial collagen fibers separated by large number of
lipid-filled macrophages tend to predispose to superficial injury.(27,28)
Chapter one: Introduction ……………………........................................................................7
2.Deep intimal injury is characterized by a split or tear that extends from the
luminal surface of a plaque deep down into the plaque substance. This type of
injury, which tends to occur in plaques that contain a large lipid-rich pool,
exposes blood to the highly thrombogenic contents of the plaque (in particular
tissue factor elaborated within the plaque, a process exacerbated by local
expression of CD40L). If sufficient thrombogenic stimulus is present the clot
may completely occlude the epicardial vessel causing acute myocardial
infarction; prevention of acute thrombotic occlusion of epicardial vessels is the
primary way by which anticoagulants (such as aspirin, clopidogrel or warfarin)
reduce the risk of recurrent myocardial infarction.(29,30)
Non-occlusive thrombosis may also cause rapid increases in the size of
atherosclerotic plaques as a result of platelet activation and elaboration of
platelet-derived growth factors (PDGF) on the surface of the plaque. Platelet
activation can directly influence the clinical course of atherosclerosis; it is
likely that acute myocardial infarction and unstable coronary syndromes are
due to thrombin and von Will brand factor-mediated platelet activation and
aggregation. Additionally, platelet activation can play a role earlier in the
pathogenesis of atherosclerosis. For example, PDGF and tumor growth factor
(TGF)-ß are two very potent mitogenic cytokines elaborated by activated
platelets that act at the site of the thrombus to promote atherosclerotic lesional
development(31,32,33).
Chapter one: Introduction ……………………........................................................................8
1.3 Inflammation and Atherosclerosis:
Atherosclerosis is an inflammatory disease of the blood vessel wall,
characterized in early stages by endothelial dysfunction, recruitment and
activation of monocyte/macrophages, dedifferentiation and migration of
vascular smooth muscle cells to form the bulk of the atherosclerotic
plaque(34,35).Furthermore, atherosclerosis is no longer considered as a disorder
due to abnormalities in lipid metabolism(36). It has been extensively
demonstrated that inflammation plays a pivotal role in atherogenesis and
mediating all stages of this disease from initiation through progression and,
ultimately, the thrombotic complications of atherosclerosis(37),.
Figure (1): Role of inflammation in atherosclerosis (38).
Chapter one: Introduction ……………………........................................................................9
1.4 Inflammatory biomarkers and their role in atherosclerosis.
Multiple cytokines and acute phase reactants have been studied having possible
parts to predict cardiovascular events in healthy men as well as for risk
stratification in established cases of CAD(39).
The only blood biomarkers currently recommended for use in cardiovascular
risk prediction by the Adult Treatment Panel are LDL cholesterol (LDL-C),
HDL cholesterol (HDL-C), and triglycerides(40).
However, plasma total cholesterol concentrations alone poorly discriminate
risk for coronary heart disease, as more than half of all vascular events occur in
individuals with below-average total cholesterol concentrations(41).
Among the most important inflammatory marker are acute-phase reactants
such as C-reactive protein (CRP),TNF-α,IL-6 serum amyloid A, and fibrinogen
provide an indirect measure of the cytokine-dependent inflammatory process in
the arterial wall .
1.4.1 Tumor necrosis factor Alpha (TNF-α):
TNF-α is a multi-functional cytokine that can regulate many cellular and
biological processes such as immune function, cell differentiation,
proliferation, apoptosis and energy metabolism(42).
TNF-α is produced by different kind of cells, including macrophages,
monocytes ,T-cells, smooth muscle cells, adipocytes , and fibroblasts (43).
It is synthesized as a 26-kDa transmembrane monomer (mTNF-α) that
undergoes photolytic cleavage by the TNF-α converting enzyme (TACE) to
yield a 17-kDa soluble TNF-α molecule (sTNF-α) (42).
Chapter one: Introduction ……………………........................................................................10
Biological responses to TNF-α are mediated by legend binding via two
structurally distinct receptors: Type I [tumor necrosis factor receptor type I
(TNF-RI); p55] and type II (TNF-RII; p75), which are present on the
membrane of all cell types except erythrocytes. These two isoforms of TNF
receptor may be found on the membrane of monocytes and T lymphocytes
(mTNFR), or circulating in the serum as a soluble receptor (sTNFR).
Soluble TNF has greater affinity for TNF-R1 than for TNF-R2.
Transmembrane TNF has equal affinity for the two receptors.TNF receptors
(TNFRs) signal as homotrimers(42,44).
TNF has a number of actions on various organ systems, generally together
with IL-1 and Interleukin-6 (IL-6):
Effect of TNF On the hypothalamus:
Stimulating of the hypothalamic-pituitary-adrenal axis by stimulating the
release of corticotrophin releasing hormone, Suppress appetite, and cause
Fever(45)
Effect of TNF On the liver:
Stimulating the acute phase response, leading to an increase in C-reactive
protein and a number of other mediators. It also induces insulin resistance by
promoting serine-phosphorylation of insulin receptor substrate-1 (IRS-1),
which impairs insulin signaling(45)
TNF is a potent chemo attractant for neutrophils, and helps them to stick to the
endothelial cells for migration, stimulates phagocytosis, and production of IL-1
oxidants and the inflammatory lipid prostaglandin E2 PGE2(45)
Chapter one: Introduction ……………………........................................................................11
A local increase in concentration of TNF will cause the cardinal signs of
Inflammation to occur: heat, swelling, redness, and pain. Whereas high
concentrations of TNF induce shock-like symptoms, the prolonged exposure to
low concentrations of TNF can result in cachexia, a wasting syndrome. This
can be found, for example, in cancer patients(46)
1.4.2 Interleukin-6 (IL-6):
It is a pleiotropic cytokine that not only affects the immune system, but also
acts in other biological systems and many physiological events in various
organs(47).IL-6 is an interleukin that acts as both a pro-inflammatory and antiinflammatory cytokine. It is secreted by T cells and macrophages to stimulate
immune response to trauma, especially burns or other tissue damage leading to
inflammation.[48]
IL-6 is one of the most important mediators of fever and of the acute phase
response. In the muscle and fatty tissue IL-6 stimulates energy mobilization
which leads to increased body temperature. IL-6 can be secreted by
macrophages in response to specific microbial molecules, referred to as
pathogen associated molecular patterns (PAMPs). These PAMPs bind to highly
important group of detection molecules of the innate immune system, called
pattern recognition receptors (PRRs), including Toll-like receptors (TLRs).
These are present on the cell surface and intracellular compartments and induce
intracellular signaling cascades that give rise to inflammatory cytokine
production.[49]
IL-6 signals through a cell-surface type I cytokine receptor complex consisting
of the ligand-binding IL-6Rα chain (CD126), and the signal-transducing
Chapter one: Introduction ……………………........................................................................12
component gp130 (also called CD130). CD130 is the common signal
transducer for several cytokines including leukemia inhibitory factor(LIF),
ciliary neurotropic factor, oncostatin M, IL-11 and cardiotrophin-1, and is
almost expressed in most tissues. In contrast, the expression of CD126 is
restricted to certain tissues. As IL-6 interacts with its receptor, it triggers the
gp130 and IL-6R proteins to form a complex, thus activating the receptor.
These complexes bring together the intracellular regions of gp130 to initiate a
signal transduction cascade through certain transcription factors, Janus kinases
(JAKs) and Signal Transducers and Activators of Transcription (STATs).(50)
IL-6
is
relevant
to
many
disease
processes
such
as
diabetes[51]
,atherosclerosis,[52] systemic lupus erythematosus[53] ,prostate cancer,[54] and
rheumatoid arthritis[55]. Advanced/metastatic cancer patients have higher levels
of IL-6 in their blood[56].
1.4.3 C-Reactive Protein:
C-reactive protein (CRP) is a circulating acute-phase reactant that is increased
many fold during the inflammatory response to tissue injury or infection. Creactive protein is synthesized primarily in the liver and its release is stimulated
by interleukin 6 (IL-6) and other pro-inflammatory cytokines. This protein has
received substantial attention in recent years as a promising biological predictor
of atherosclerotic disease(57).
There is an evidence that CRP may play a direct role in the pathogenesis of
atherosclerosis. Zwaka T.P. et al. (2001) demonstrated that CRP is markedly
up-regulated in atheromatous plaques, where it may promote LDL- C uptake by
macrophages, a key step in atherogenesis(58).
Chapter one: Introduction ……………………........................................................................13
C-reactive protein may also induce the expression of intercellular adhesion
molecules by endothelial cells, thereby facilitating the recruitment of
circulating monocytes to plaque sites, and can bind to and activate complement
in serum(59,60).
These effects appear to be mediated through CRP-induced secretion of
Endothelin -1 and IL-6(61) .
CRP has been proposed to be an independent risk factor for atherosclerosis
including ischemic heart disease, restenosis after percutaneous coronary
intervention, stroke, and peripheral artery disease (62,63). Some studies showed
CRP is a better predictor than LDL(64,65).
The circulating value of CRP is much more accurately reflects on-going
inflammation than do other biochemical parameters of inflammation, such as
plasma viscosity or the erythrocyte sedimentation rate. This is because of the
plasma half-life of CRP is the same (about 19 h) under all conditions, and the
sole determinant of the plasma concentration is therefore the synthesis rate,
which reflects the intensity of the pathological process stimulating CRP
production (66).
Levels of CRP associated with vascular inflammation are much lower than
those previously considered normal. Therefore assays, with the ability to
measure lower concentrations of CRP, known as high-sensitivity CRP (hsCRP) or as Cardio CRP (CCRP), were developed to achieve the required
sensitivity. Furthermore Physicians have become accustomed to use the "highsensitivity CRP (hsCRP)" terminology when considering measurement of CRP
for vascular disease risk stratification (67).
Based on many epidemiological data, it has been proposed to categorize
patients according to their baseline hs-CRP levels into:
Chapter one: Introduction ……………………........................................................................14
-A low risk (hsCRP < 1 mg/L),
-An intermediate risk (hsCRP between 1 and 3 mg/L) and
-A high risk group (hsCRP > 3 mg/L) (68).
Furthermore, an immune histological study showed that hsCRP is present in
human coronary athermanous lesions and its level positively correlates with the
degree of progression of atherosclerosis(69).
CRP is a proarthrogenic factor throughout the entire spectrum of this process,
ranging from fatty streak formation to atherothrombosis and tissue injury of
ischemic regions; for example, CRP has been shown to modulate endothelial
cell functions and increase Smooth muscle cell migration and mediate the
uptake of oxLDL by macrophages. CRP itself is a chemo attractant factor for
monocytes and induces the production of
MCP-1 and expression of adhesion
molecules on endothelial cells(67, 70).
1.5 Oxidative Stress :
Oxidative stress can be defined as an imbalance develops between the
production of ROS and the efficacy of the cell's antioxidant defense, leading to
an altered redox status which can contribute to endothelial dysfunction and/or
cell death(71,72).
1.5.1 Causes of oxidative stress:
It occurs as result of several factors, including:
1.Exposure to cigarette smoking, alcohol, infection, toxins, ionizing radiation,
UV light, environmental pollutants & xenobiotics (73) .
2.Strenuouse physical activity.(74)
Chapter one: Introduction ……………………........................................................................15
3.Diabetes mellitus, hyperlipidimia, ischemia-reperfusion, hypertension,
&aging (75,76)
4.Hormons such as (insulin & leptin),(75) Pro-inflammatory cytokine like TNFα & IL-6(77),and Growth factors like platelet derived growth factor, epidermal
growth factor &vascular endothelial growth factor (78).
1.5.2 Free radicals:
Free radicals can be defined as molecules or molecular fragments containing
one or more unpaired electrons. The presence of unpaired electrons usually
confers a considerable degree of reactivity upon a free radical. Those radicals,
derived from oxygen, represent the most important class of such species
generated in living systems(79).
These free radicals are highly reactive, unstable molecules react with (oxidize)
various cellular components including DNA, proteins, lipids / fatty acids and
advanced glycation end products (e.g. carbonyls) which lead to DNA damage,
mitochondrial malfunction, cell membrane damage and eventually cell death
(apoptosis - which is the term for programmed cell death)(80).
Free radicals are generally reactive oxygen or nitrogen species. Examples of
free radicals (oxidizing molecules) are hydrogen peroxide, hydroxyl radical,
nitric oxide, peroxynitrite, singlet oxygen, superoxide anion and peroxyl
radical.(81) Superoxide is generated via several cellular oxidase systems
(enzyme reactions). Once formed, it participates in several reactions yielding
various free radicals such as hydrogen peroxide, peroxynitrite, etc. In turn,
these can lead to chain reaction byproducts that also act to damage cells
(example: lipid peroxidation products)(81).
Chapter one: Introduction ……………………........................................................................16
An example of a very potent free radical is peroxynitrite which is 1000X more
potent as an oxidizing compound than hydrogen peroxide. Markers of
peroxynitrite formation (such as nitrotyrosines or isoprostanes) can be found in
many disease states including Alzheimer’s brains, Parkinson’s disease, chronic
heart disease, liver disease, areas of inflammation, and so on. Thus, excess free
radical formation is associated with many disease states. Inflammation, poor
blood flow, degenerative diseases, and toxin exposures among other
mechanisms all lead to oxidative stress.(82)
1.5.3 Antioxidants :
Antioxidants are
any substance (molecule, compound and enzyme) that
significantly counteract the oxidants. Most antioxidants are electron donors and
react with the free radicals to form innocuous end products such as water.
Thus, antioxidants protect against oxidative stress and prevent damage to cells.
There are many examples of antioxidants(83):
1.Enzymatic antioxidants like: superoxide dismutase, Catalase & glutathione
peroxidase,
2.Endogenous molecules like : glutathione (GSH), alpha lipoic acid
3.Essential nutrients like : vitamin C, vitamin E, selenium, N-acetyl cysteine.
TNF-α impairs GSH production by several mechanisms, resulting in lower
GSH levels. Furthermore, oxidative stress increases TNF alpha production.
Consequently GSH disturbances and enhanced TNF alpha production /
activation lead to a pathogenic "loop" or vicious cycle (84,85).
Chapter one: Introduction ……………………........................................................................17
1.5.3 Oxidative stress in atherosclerosis:
Oxidative stress has been identified throughout the process of atherogenesis,
beginning at the early stage when endothelial dysfunction is barely apparent(86).
As the process of atherogenesis proceeds, inflammatory cells, as well as other
constituents of the atherosclerotic plaque release large amounts of ROS, which
further facilitate atherogenesis. In general, increased production of ROS may
affect four fundamental mechanisms that contribute to atherogenesis: oxidation
of LDL, endothelial cell dysfunction, vascular SMCs, growth and monocytes
migration(87).
A number of studies suggest that ROS oxidize lipids and that the oxidatively
modified LDL is a more potent proatherosclerotic mediator than the native
unmodified LDL(88).
The suggestion is based on the observations that high plasma levels of ox-LDL
are present in patients with atherosclerosis and that antibody to ox-LDL is
detected in plasma of most patients with atherosclerosis (89).
Strong evidence in favor of a pro-atherosclerotic role for ox-LDL comes from
a number of studies demonstrating the noxious effects of ox-LDL on various
components of the arterial wall. For example, ox-LDL causes activation of the
endothelial cells lining the arterial wall, resulting in the expression of several
adhesion molecules that facilitate the adhesion of monocytes/macrophages (90).
ox-LDL also activates inflammatory cells and facilitates the release of a
number of growth factors from monocytes/macrophages (91,92).
Chapter one: Introduction ……………………........................................................................18
Vascular SMCs exhibit intense proliferation when exposed to ox-LDL. oxLDL enhances the formation of matrix metalloproteinase (MMPs) in vascular
endothelial cells and fibroblasts (93) thus setting the stage wherein oxidative
stress leads to rupture of a soft plaque. In addition, ox-LDL up regulates the
expression of its endothelial receptor LOX-1 and other scavenger receptors
mainly expressed on macrophages/monocytes. The increased expression of
these receptors is responsible for the uptake of ox-LDL and the formation of
foam cells, which is an early step in atherogenesis(80).
1.6 Treatment of Atherosclerosis:
The treatment of atherosclerosis relies heavily on the reduction of risk
factors particularly elevated serum lipids level, hypertension, diabetes and
cigarette smoking. The incidence of cardiac and cerebral infarction can be
diminished considerably by controlling risk factors. Lifestyle factors are also of
greatest importance in the treatment of atherosclerosis(94).
1.6.1 Life style change:
The most common treatments focus on lifestyle changes to reduce cholesterol
and other problems that contribute to atherosclerosis. Dietary modifications
usually incorporate eating foods that are low in saturated fats, cholesterol, sugar,
and animal proteins. Foods high in fiber, such as fresh fruits and vegetables, and
whole grains, are encouraged. By consuming fruits and vegetables, the person
also consumes helpful dietary antioxidants, such as carotenoids found in
vegetable pigments, and bioflavonoid in fruit pigments. Liberal use of onions
and garlic is recommended, as well as eating fish, especially cold-water fish,
Chapter one: Introduction ……………………........................................................................19
such as salmon. Smoking, alcohol, and coffee are to be avoided, and exercise is
strongly recommended(95).
1.6.2 Medical Treatment:
1.6.2.1 Lipids modifying drugs:
A. HMG-CoA reductase inhibitors ( Pravastatin, Lovastatin ,Fluvastatin,
Atorvastatin, Rosuvastatin):
These agents are competitive inhibitors of 3-hydroxy-3-methyl Co-A reductase,
an enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis,
resulting in up-regulation of LDL receptors in response to the decrease in
intracellular cholesterol(96).
B. Fibric acid derivatives(Fenofibrate, Gemfibrozil ):
They increase the activity of lipoprotein lipase and enhance the catabolism of
triglyceride-rich lipoproteins, which is responsible for decrease in triglyceride
levels by 20-50% and increase HDL cholesterol levels by 10-15%(97).
C. Bile acid sequestrants(Cholestyramine, Colestipol):
The bile acid sequestrants block enterohepatic circulation of bile acids and
increase the fecal loss of cholesterol. This results in a decrease in intrahepatic
levels of cholesterol. The liver compensates by up-regulating hepatocyte LDL
receptor activity. The net effect is a 10-25% reduction in LDL cholesterol, but
no consistent effect on triglycerides or HDL cholesterol exists(98).
Chapter one: Introduction ……………………........................................................................20
D. Cholesterol absorption inhibitors :
Ezetimibe is The first drug of this class is approved by the FDA in November
2002. It interferes with the absorption of cholesterol and when used as
monotherapy, reduces LDL-C by 15–20% (98).
E. Cholesteryl Ester Transfer Protein inhibition (CETPi).
The CETP inhibitors anacetrapib, dalcetrapib and torcetrapib were shown
to bind to CETP, resulting in the formation of a stable complex between CETP
and HDL at concentrations relevant to their inhibition of neutral lipid transfer.
This therefore appears to be a key mechanism whereby these agents block
CETP activity. CETP, LCAT (lecithin-cholesterol acyltransferase)and PLTP
(phosphor-lipid transfer protein) play important roles in lipoprotein remodeling
in plasma. CETP inhibitory activity has also been
shown to decrease
formation of pre-beta HDL. These lipid-free apoA-I or discoidal HDL
particles, are the preferred acceptors of ABCA1-dependent cholesterol efflux,
an important step in reverse cholesterol transport(99).
F. Phospholipase A2 inhibitors.
The secreted PLA2s and the lipoprotein-associated phospholipase A2 (LpPLA2) have been associated with atherogenesis and its complications. These
two enzymes produce biologically active metabolites that are involved in
several phases of the atherosclerosis process. Therefore, inhibitors of these
enzymes like darapladib and varespladib emerge as promising therapeutical
options for treating patients with atherosclerosis(100).
Chapter one: Introduction ……………………........................................................................21
1.6.2.2Antioxidant medications:
Epidemiologic studies have demonstrated an association between increased
intakes of antioxidant vitamins, including vitamins C, E, and beta-carotene, and
reduced mortality from coronary artery disease. Antioxidants are thought to
reduce the oxidation of LDL cholesterol, inhibit lipid accumulation in the
arterial wall and improve endothelial function (101).
1.7 New anti-inflammatory therapeutic approaches:
Inflammation contributes to the formation and progression of atherosclerosis
and the therapeutic potential of some anti-inflammatory drugs have been
evaluated for possible anti atherosclerotic activity. Recent findings suggest that
some agents with anti-inflammatory properties appear to have beneficial
effects on atherosclerosis or subsequent risk for cardiovascular events, while
others have been disappointing(102).
*Angiotensin-converting enzyme inhibitors and angiotensin receptor
blockers have been shown to inhibit the inflammatory effects of angiotensin II
through modulation of pro-inflammatory cytokines, NF-KB, oxidative stress,
and nitric oxide synthesis
(103)
. Fukuda et al (2009) suggest that inhibition of
rennin– angiotensin system attenuates periadventitial inflammation and reduces
atherosclerotic lesion formation(104).
*(Pioglitazone and rosiglitazone) peroxisome proliferator activator
receptor PPAR-alpha and gamma have demonstrated anti-inflammatory
properties. PPAR-gamma agonists have been shown to reduce proinflammatory mediators like NF-KB, to inhibit the expression of VCAM-1 and
Chapter one: Introduction ……………………........................................................................22
ICAM-1 in activated endothelial cells and significantly decrease the homing of
macrophages and monocytes to atherosclerotic plaques.(105,106).
*Nonsteroidal
anti-inflammatory
drugs
(NSAID):
selective
inhibition of the COX-2 enzyme with celecoxib prevents the development of
atherosclerotic lesions in the proximal aortas from Apo E-/- mice through
reduction in expression of ICAM and VCAM(107).
*Tumor necrosis factor-[alpha] blocking agents(Etanercept)
Anti-TNF therapy can improve other risk factors for accelerated
atherosclerosis, including promoting a decrease in insulin resistance, CRP, IL6, and an increase in HDL-C(108,109). A recent study from Sweden suggested
that the risk for developing first cardiovascular events in rheumatoid arthritis is
lower in patients treated with TNF blockers (110).
1.8 Some Aspects of the drugs used in this study.
1.8. Glimepiride:
It is one of the third generation sulphonylurea drugs useful for control of
diabetes mellitus type 2.(111)
Preclinical investigations of glimepiride suggested a number of potential
benefits over sulfonylurea currently available including lower dosage, rapid
onset, longer duration of action and lower insulin C-peptide levels, possibly
due to less stimulation of insulin secretion and more pronounced extra
pancreatic effects .(112)
Chapter one: Introduction ……………………........................................................................23
1.8.1.1 Mechanism of action:
The primary mechanism of action of glimepiride in lowering blood glucose
appears to be dependent on stimulating the release of insulin from functioning
pancreatic beta cells. In addition, extra-pancreatic effects may also play a role
in the activity of glimepiride.(113)
1.8.1.2 Pharmacological actions:
Glimepiride administration can lead to increased sensitivity of peripheral
tissues to insulin. These findings are consistent with the results of a long-term,
randomized, placebo-controlled trial in which AMARYL® (glimepiride)
therapy improved postprandial insulin/C-peptide responses and overall
glycemic control without producing clinically meaningful increases in fasting
insulin/C-peptide levels.(113)
Glimepiride also has been reported to show extra pancreatic actions including
phosphorylation of insulin receptor substrate-1 (IRS-1), an upstream regulator
of phosphatidylinositol 3-kinase (PI3-kinase),in adipocytes(114).
Treatment of diabetic patients with conventional sulfonylurea such as
glibenclamide has been reported to be associated with adverse cardiovascular
effects and a high incidence of cardiovascular death (115).
Previous studies have shown that glimepiride has less adverse cardiovascular
activity than conventional sulfonylurea and does not appear to have deleterious
effects on ischemic preconditioning (116,117).
Chapter one: Introduction ……………………........................................................................24
Glimepiride
administered once daily, has been demonstrated to offer
therapeutic advantages over other sulfonylureas in terms of glucose leveldependent insulintropic action, insulin-sparing effects, and hypoglycemic risk
(118-120)
. Furthermore, glimepiride exhibits inhibitory effects on human platelets
aggregation through selective suppression of the cyclooxygenase pathway,
suggesting that it may have therapeutic potential in diabetic patients with
enhanced platelet function (121).
In addition, there are several reports showing beneficial effects of sulfonylurea
against development of cardiovascular disease in rabbits fed atherogenic diet
as well as in hyperglycemic patients (122).
Recently, glimepiride has been demonstrated to inhibit the formation of
atheromatous plaques in thoracic aorta of high-cholesterol fed rabbits .The
mechanism by which glimepiride induces atheroprotective effects remains to
be elucidated(123).
1.8.2 Repaglinide:
It is a new hypoglycemic agent, a member of the carbamoylmethyl benzoic
acid family.
1.8.2.1 Mechanism of action
Its mechanism of action is partly similar to that of sulfonylurea. Repaglinide
stimulates the release of insulin from pancreatic beta-cells by inhibition of
potassium efflux resulting in closure of ATP regulated K+ channels (124,125)
This results in depolarization of the cell and subsequent opening of calcium
channels, leading to influx of calcium into the cells which causes release of
Chapter one: Introduction ……………………........................................................................25
insulin
(126).
However, repaglinide regulates these channels via a different
binding site than that of sulfonylurea.
1.8.2.2 Pharmacological actions
Repaglinide improves glycemic control through a physiological action on the
reduced phase of insulin secretion in type 2 diabetes mellitus(127).
Some results from the literature demonstrate that repaglinide has a favorable
effect on the parameters of antioxidative balance. It significantly improved the
activity of superoxide dismutase and diminished lipid peroxidation in the
serum of type 2 diabetic patients(128,130).
it is possible that the drug itself, being a benzoic acid derivative, has some
antioxidant activity(128) .
1.8.2.3 Pharmacokinetics
Repaglinide is rapidly absorbed from the GI tract and reaches maxim um
plasma concentration approximately 1 hour after ingestion when given as a
tablet. (129,130)
The bioavailability of the oral formulation was found to be 63% in a study
comparing 2mg (as a tablet) with the same dose given IV to healthy
volunteers(129). The elimination half life is short, about 1 hour(130).
Repaglinide is extensively metabolized in the liver with only a very small
proportion of the unchanged drug appearing in the urine (128,129).
Its metabolites do not contribute to the glucose-lowering effect of the drug.
The major metabolites of repaglinide are excreted mainly into the bile with
Chapter one: Introduction ……………………........................................................................26
90% of the dose being recovered in the faces with less than 2% representing
the parent drug(131,132).
Concurrent administration with food did not appear to alter the absorption of
repaglinide significantly in healthy volunteers (133).
It has a fast onset and a relatively short duration of action, allowing treatment
to be taken flexibly with meals(134).
Repaglinide stimulates glucose-driven pancreatic insulin secretion and
facilitates postprandial insulin response, thus lowering the risk of
hypoglycemia (135).
Repaglinide is well tolerated and has efficacy at least equivalent to comparator
sulphonylurea , but with a significant 60% risk reduction for severe
hypoglycemia (136).
1.8.2.4 Adverse effects :
The tolerability profile of repaglinide appears to be similar to sulphonylurea
drugs, however data are still limited. In trials the most frequently reported
adverse events were hypoglycemia (16%), upper respiratory tract infections
(10%), rhinitis (7%), back pain (6%), diarrhea (4%), nausea (3-5%),
constipation (3%), arthralgia (3- 6%), headache (5%) and sinusitis (36%)(130,131).
There is also the potential for weight gain(137). There have been isolated reports
of increases in liver enzymes most cases were mild and transient.
Hypersensitivity reactions can occur as itching, rashes and urticaria. There is
no reason to suspect cross-allergenicity with sulphonylurea (138).
Chapter one: Introduction ……………………........................................................................27
1.8.2.5 Contraindications :
Hypersensitivity to repaglinide. Type 1 diabetes Diabetic ketoacidosis with or
without coma Pregnancy and lactation. Children under 12 years of age. Severe
renal or hepatic impairment Concomitant therapy with medicinal products
inhibiting or inducing cytochrome P450 3A4(138).
1.9 Therapeutic approach:
A major consequence of type 2 diabetes is accelerate onset of atherosclerosis
which an important risk factor in the development of ischemic cardiovascular
disease. Glimepiride is a common drug used for treatment of type 2 diabetes, a
very little studies show that glimepiride will reduce atherosclerosis progression
in high cholesterol fed rabbits (123). so in this study we evaluate the protective
effect of Glimepiride in atherosclerosis progression as atherosclerosis is
induced by oxidative &inflammatory process.
Repaglinide is also a common drug used for treatment of type 2 diabetes ,also
some studies show that repaglinide have antioxidative effects (128),so in this
study we also evaluate the protective effect of repaglinide in atherosclerosis
progression as atherosclerosis is induced by oxidative &inflammatory process.
Chapter one: Introduction ……………………........................................................................28
1.10 Aim of the study:
The main aim of the present study is to assess the effect of Glimepiride and
Repaglinide on progression of atherosclerosis in hypercholesterolemic rabbits.