Atherosclerosis

Stages involved in plaque formation

1. Endothelial cell injury, resulting in endothelial dysfunction 2. Endothelial activation 3. Accumulation of lipoproteins 4. Platelet adhesion 5. Wbc recruitment 6. Adaptive immune response 7. Smooth muscle cell recruitment

Endothelial cell injury

1. Endothelial cell injury, resulting in endothelial dysfunction. Injury can be caused by hyperlipidaemia/hypercholesterolaemia, hypertension & its associated haemodynamic turbulence, viruses, toxins (e.g. from cigarette smoke) or irradiation. Toxins and irradiation promote the formation of harmful ROS. The damaged endothelium is usually morphologically intact but develops an activated pro-atherogenic phenotype. People with conditions associated with endothelial dysfunction (e.g. diabetes) are thus more susceptible to developing atherosclerosis.

Endothelial activation

2. Endothelial activation results in increased expression of adhesion molecules (CAMs), increased endothelial permeability and release of inflammatory cytokines and ROS. VCAM-1 is an example of a cell adhesion molecule and is a key marker of endothelial dysfunction.

Platelet adhesion

4. Platelet adhesion to sub-endothelium via collagen receptors. Platelets facilitate the recruitment of inflammatory cells towards lesions, release inflammatory mediators, contribute to endothelial dysfunction and determine whether plaque will rupture.

SMC recruitment

7. Smooth muscle cell recruitment for fibroproliferative response SMCs are activated by signalling (PDGF, TNF-alpha, IL-1, TGF-beta) from activated platelets, macrophages, foam cells and endothelial cells. They migrate into intima across the damaged internal elastic lamina, proliferate and secrete ECM macromolecules (e.g. collagen, elastic fibres, proteoglycans) to form a fibrous cap around the lipid-rich plaque core. This converts fatty streak into a more stable FIBRO-FATTY PLAQUE (atheroma - mature atherosclerotic plaque) that progressively grows. The core of this becomes necrotic with cholesterol-crystal-filled clefts as foam cells die. The fibrous cap maintains structural integrity and hardening of the plaque but can be compromised by prolonged pro-inflammatory signals recruiting immune cells, leading to plaque rupture and further pathology.

Adaptive immune response

Adhesion molecules on endothelial cells attract DCs and T cells into tunica intima. DCs take up apolipoprotein from lipoproteins and migrate to lymph nodes, where they present ApoB antigens for naive T cell activation. T cells differentiate into effector cells and form Th cells (among other cell types), which help to drive atherosclerosis - promote collagen formation, secretion of chemokines and pro-inflammatory cytokines, such as IFN-γ. IFN-γ promotes ROS generation to further increase local [mLDL] and matrix metalloproteinase (MMP) secretion for thinning of fibrous cap. It also causes macrophages to become M1 polarised, promoting inflammation.

Decreasing activation of T cells for anti-atherogenic therapy

Alternatively, decreasing the activation of other T effector cells could be a future anti-atherogenic therapy. One study by Stroes et al. (2010) compared atherosclerotic plaques in patients who received mycophenolate mofetil (immunosuppressant drug) vs placebo. This drug can reduce activation of macrophages, DCs, T cells and NKs and increase Treg production and the study found that the percentage of CD3+ T cells was lower in plaques from subjects who received MMF, while their percentage of Treg cells was higher. This shows that immunosuppressive treatments affecting T cells could be a future treatment for atherosclerosis. Further research is definitely needed, however, especially considering this study only investigated 21 patients and gave MMF for a limited time only (2 weeks).

Clinical consequences of plaques - angina and intermittent claudication

Although atherosclerosis itself is asymptomatic, the pathological changes it induces in the vasculature provoke chronic and acute complications. As a stable atherosclerotic lesion grows, arterial lumen diameter is decreased. This reduces downstream blood flow, causing tissue ischaemia, which is worsened during activity when O2 demand is higher. This underlies stable angina pectoris - chest pain from myocardial hypoxia from blockage in the coronary circulation, usually brought on by exertion and reverses upon rest. If atherosclerosis occurs peripherally, ischaemia in the vessels of the limbs causes peripheral vascular disease, which is characterised by intermittent claudication - muscle pain/aches/cramps/fatigue upon mild exertion (classically within the calf muscle) due to arterial insufficiency from reduced lumen diameter. Excessive limb ischaemia can lead to necrosis and gangrene.

Animal models of atherosclerosis

Animal models of atherosclerosis have contributed greatly to our understanding of the pathogenesis and prevention of the disease. Apolipoprotein E deficient (ApoE -/-) and LDL receptor deficient (LDLR -/-) mice are the most commonly used models. KO mice are necessary because WT mice are resistant to atherosclerosis and require long periods on high fat diets (~1 year) before plaques begin to develop. ApoE is a component of lipoproteins that interacts with the LDLR on peripheral cells to facilitate lipoprotein uptake. Deficiency of these proteins thus promotes LP accumulation in the circulation, leading to high plasma [LDL]. ApoE -/- mice are the most popular animal model because they develop spontaneous atherosclerosis on normal diets and their lesions closely resemble those in humans. Plasma cholesterol levels can be increased by feeding a Western type diet. However, ApoE KO fundamentally changes macrophage activity, which worsens plaque formation. LDLR -/- mice require high-fat, high-cholesterol diets to develop atherosclerosis but are becoming more popular as they are good models for familial hypercholesterolaemia. Both knockout mice present with hypercholesterolaemia, which can be worsened by a high-caloric diet, resembling that of the western human population. These mice develop atherosclerosis due to the hypercholesterolaemia. These models have enabled the association of individual genes with atherogenesis.

Anti-thrombotic drugs

Anti-thrombotic drugs include antiplatelet agents (e.g. aspirin, abciximab) and thrombolytic agents (e.g. streptokinase, alteplase). Antiplatelet drugs interfere with platelet activity and are mainly used in the treatment & prevention of arterial thrombosis. Aspirin is a non-competitive COX1/2 inhibitor which inhibits TXA2 mediated activation of new platelets and their aggregation, reducing the likelihood that a clot will form. Abciximab is a platelet glycoprotein GPIIb/IIIa receptor inhibitor that prevents platelets from activating upon contact with fibrinogen or von Willebrand factor. This prevents platelet adhesion, aggregation and formation of platelet plug in early haemostasis. These drugs are often taken after an initial cv event to prevent further pathology. Thrombolytics dissolve existing thrombi by upregulating fibrinolysis. They are used clinically for rapid reversal of thrombotic arterial occlusion in acute MI and thrombotic stroke. Streptokinase stimulates conversion of plasminogen to plasmin to accelerate haemolysis (dissolving of blood clots). Alteplase is a recombinant tPA, which increases tPA levels to convert plasminogen to plasmin, accelerating haemolysis like streptokinase. Although this is much more expensive than streptokinase, clinical trials show a slight increase in efficacy compared to streptokinase (GUSTO trial).

Anticoagulants

Anticoagulants (e.g. heparin) inhibit the coagulation cascade and thus prevent fibrin deposition. Heparin stabilises antithrombin-III interaction with key coagulation enzyme factors (especially factor Xa and thrombin (IIa)) to decrease thrombin formation, thereby downregulating the coagulation cascade. It is administered intravenously in the acute treatment of deep vein thrombosis, pulmonary embolism and arterial thromboembolism. However, it does have the potential to cause some serious side effects, including haemorrhage and thrombocytopenia.

Antihypertensive drugs

Antihypertensive drugs can help to reduce bp. There are several different types, including Ca2+ channel antagonists, thiazide diuretics and beta blockers.

What is atherogenesis?

Atherogenesis is the formation and development of an atheroma (subendothelial plaque), which occurs over a long period of time (decades). It is currently thought that atherogenesis arises in response to injury to the arterial wall (response to injury hypothesis, Ross 1999) and is viewed as an unusual form of chronic inflammation.

Clinical consequences of plaques - aneurysm

Atherosclerosis can also result in an aneurysm. The plaque causes artery wall to weaken due to plaque pressure on the tunica media, promoting atrophy and loss of smooth muscle & elastin. The fragile artery wall thus expands/balloons outwards, forming an aneurysm which may burst and lead to serious haemorrhage - for example, AAA rupture producing bleeding into peritoneal cavity.

Atherosclerosis overview

Atherosclerosis is a chronic, inflammatory disease of the arterial wall characterised by the formation of fatty atheromatous plaques in the tunica intima of large arteries, often at bifurcations where flow is turbulent. It is of great clinical importance as the disease process can have serious consequences, including heart attacks and stroke. It is the underlying cause of most cv disease and so is responsible for much morbidity and mortality in developed countries. Preventing and treating atherosclerosis is therefore very important. Plaque is formed from modified lipids and leukocytes in a dynamic process. It becomes increasingly complex as the disease progresses and, depending on its composition and location, will have varying clinical consequences.

Cholesterol transport

Cholesterol is transported in the blood by lipoproteins, which have apolipoproteins on their outer surface. There are several different types of lipoproteins that transport cholesterol - HDL (high density lipoprotein), LDL (low), IDL (intermediate), VLDL (very low) & chylomicrons (in order of decreasing density). VLDL, IDL and LDL are pro-atherogenic. HDL is anti-atherogenic - reverse cholesterol transport and anti-oxidant properties. LDLs ('bad') deliver cholesterol to cells. HDLs ('good') deliver cholesterol to the liver to be excreted. Raised LDL-cholesterol levels increase risk of developing atherosclerosis as there is more cholesterol circulating that can be deposited in artery walls - cannot be taken up by peripheral cells. Similarly, low plasma HDL-cholesterol is associated with increased risk of cv disease. Population studies have demonstrated that elevated levels of LDL cholesterol and apolipoprotein B (apoB) 100, the main structural protein of LDL, are directly associated with risk for atherosclerotic Cv events.

What scientific approaches have told us the most about the mechanisms of development of vascular disease?

Clinical trials and epidemiological studies Genetic and experimental approaches Discuss merits and limitations For Atherosclerosis and VTE

How can endothelial dysfunction be measured?

Endothelial dysfunction can be measured by IV infusion of ACh and observing changes in the tone of target vessels with angiography. This was first demonstrated by Ludmer et al. (1986). In normal arteries, vasodilation occurs because ACh acts on endothelial cells, stimulating production of NO. However, in atherosclerotic arteries, the damaged endothelium prevents ACh from binding and stimulating NO production. Instead, it binds to M3 Gq-coupled receptors on VSMCs, causing vasoconstriction.

Evidence that MCP1 is involved in atherosclerosis

Experiments by Boring et al. (1998) provided evidence that MCP1 is involved in atherosclerosis. Aortic root sections were taken from ApoE KO mice and KO mice lacking the MCP1 receptor (CCR2) and ApoE. Under the fluorescence microscope with a macrophage-specific fluorescent antibody, they observed decreased macrophages in the lesion area and decreased lesion formation in the CCR2-/- ApoE -/- mice compared to ApoE-/- mice. This suggests that macrophage chemokines are critical in their recruitment and atherogenesis.

Renard et al (2004) - risk factors that induce atherosclerosis in mice

Experiments by Renard et al. (2004) demonstrated the role of some of these risk factors in inducing atherosclerosis in mice. Diabetes was first induced in LDLR KO mice using virally-induced autoimmune destruction of pancreatic islet cells. On a cholesterol-rich diet, mice developed advanced lesions with intralesional haemorrhage, suggesting that cholesterol plays a role in atherogenesis. However, on a cholesterol-free diet, these mice still had accelerated lesion initiation and increased arterial macrophage accumulation. When treated with insulin, the size of lesions decreased, suggesting diabetes is also a risk factor for plaque development.

Limitations of atherosclerosis animal models

However, there are several disadvantages to using mice to model atherosclerosis: Mice plaques do not rupture - unless on extremely high lipid diet for a very long time It is difficult to study plaques in mice coronary vessels - they are too small Atherosclerosis takes approximately 40 years to develop in humans. In animal models, atherogenesis is accelerated, which may influence the atherogenic mechanism.

Future directions for atherosclerosis treatment

However, while these therapies help to reduce atherosclerotic mortality and morbidity, there is little currently that can interfere with the actual disease process. Several drugs to block the chronic inflammatory mechanism have been theorised, including canakinumab (anti-IL-1β) and CB2 (cannabinoid 2) receptor agonists. Zhao et al. (2010) tested the cannabinoid WIN55212-2 (CB2R agonist) in ApoE -/- mice on a high-fat diet and found it reduced lesion size, macrophage content and VCAM-1 expression, demonstrating its anti-atherosclerotic effects in an animal model. These effects were almost fully abolished by AM630 (CB2 antagonist), confirming that the beneficial effects of WIN55212-2 may be mediated through the CB2 receptor. A tolerogenic vaccine has also been hypothesised which induces the action of atheroprotective Tregs against atherosclerosis-related antigens to suppress the effector mechanisms of Th1 cells which enhance M1 macrophage differentiation. However, one concern with this is the potential for Tregs to switch to a pathogenic phenotype.

Role of IFN-gamma in atherosclerosis + EE

IFN-γ promotes ROS generation to further increase local [mLDL] and matrix metalloproteinase (MMP) secretion for thinning of fibrous cap. It also causes macrophages to become M1 polarised, promoting inflammation. Its role in atherogenesis is demonstrated by IFNγ KO mice, which exhibit smaller atherosclerotic lesions and a 60% reduction in lesion lipid accumulation (Gupta 1997). Buono et al. (2003) also showed that injection of recombinant IFN-γ results in lesion growth and an increase in their size.

Evidence of link between modifiable risk factors and atherosclerosis in humans

In humans, the association between these RFs and atherosclerosis came from several prospective, longitudinal cohort studies into cv disease. The Framingham Heart Study, which began in 1948, recruited thousands of men and women for a prospective study into risk factors for coronary heart disease. Every 2 years, a physical examination (bp, blood lipid concs) and lifestyle interviews (smoking, alcohol, lifestyle) were performed to gather data. Some of its key results to date include identifying smoking, hypertension, cholesterol level and obesity as RFs for heart disease. It also showed that exercise and high HDL cholesterol decrease risk. The Multiple Risk Factor Intervention Trial (MRFIT) is another prospective study of ~360,000 middle-aged men who were followed for 16 years. It found a positive correlation between cholesterol levels and relative risk of mortality and also reinforced the importance of multiple risk factors contributing to CHD. The benefits of low risk behaviours, such as not smoking and drinking alcohol, were also demonstrated.

Accumulation of lipoproteins

Increased endothelial permeability facilitates entry of circulating lipoproteins (LDL and VLDL) into the tunica intima. They accumulate here by binding to proteoglycans in the ECM. LDLs then undergo chemical alterations mediated by ROS and pro-oxidant enzymes released from endothelial cells, SMCs or macrophages, forming modified/oxidised LDLs. These are pro-inflammatory, promoting wbc recruitment & foam cell formation, as well as preventing LDL from being able to exit intima and re-enter the circulation.

Non-modifiable risk factors for atherosclerosis

Non-modifiable risk factors include: Male sex - as oestrogen is thought to have a protective effect by stimulating endothelial NO production for vasodilation. Menopause - as oestrogen's protective effect is lost Increasing age - due to progressive stiffening of vessel walls, elastin breakdown, calcification and increased likelihood of hypertension. Family history of CHD and genetic factors - e.g. FHH (earlier card)

What lines of evidence link cholesterol to atherogenesis?

Lines of evidence linking cholesterol to atherogenesis: Classic prospective studies - Framingham and MRFIT Mendelian genetics - LDLR and PCSK9 & familial hypercholesterolaemia Mouse models of atherogenesis - ApoE -/- and Ldlr -/- Randomised clinical trials of statins - 4S study

Prevention and therapies to limit atherosclerosis

Preventative measures can be taken to reduce your modifiable risk factors to limit atherosclerosis-mediated disease. This includes general lifestyle measures, such as diet changes (less saturated fat & cholesterol), stopping smoking, weight loss & increasing exercise, as well as pharmacological strategies. Drugs: antihypertensives, statins & other cholesterol lowering drugs (bile acid sequestrates, anti PCSK9 antibodies)

2 types of macrophages and their roles in atherosclerosis

Macrophages can be classified into two types - classically activated, pro-inflammatory M1 cells and alternatively activated, anti-inflammatory M2 cells. As atherosclerotic lesions progress, there is a shift from M2 to M1 macrophages, promoting further inflammation. This shift suggests that the balance between M1 and M2 cells is involved in determining the formation and regression of plaques and so reducing the number of M1 cells/increasing the number of M2 cells could represent a therapeutic target for atherosclerosis. Khallou-Laschet et al. (2010) found that, regardless of atherosclerosis stage, the prevalence of M2 phenotype was associated with smaller plaque surface areas in ApoE KO mice, demonstrating their useful anti-atherogenic role. Moore et al. (2013) have since suggested induced M2 polarisation as a potential treatment to promote an anti-inflammatory, anti-atherogenic state. However, it is important to note that, due to the complexity of inflammatory stimuli present in the plaque, the terms M1 and M2 are probably an oversimplification. It is likely that there are a range of overlapping phenotypes in atherosclerotic lesions.

Wbc recruitment

Monocytes and then T cells are recruited to the arterial wall. Monocytes adhere to apical surface of endothelium by interacting with endothelial CAMs, including VCAM-1, ICAM-1 and selectins. These have been upregulated on the activated endothelium by ROS, OxLDL and cytokines. They then migrate into subendothelial space by chemotaxis in response to chemoattractants, such as monocyte-chemoattractant protein 1 (MCP1/CCL2). Next, monocytes differentiate into activated macrophages, which have several roles: Secrete cytokines (TNF-alpha, IL-1) that promote further wbc recruitment and proliferation Produce toxic O2 species, which cause further LDL oxidation Engulf oxidised LDL and cholesterol crystals through scavenger receptors, forming foam cells, which aggregate in blood vessel wall. LDLs cannot be taken up by the traditional LDL receptors because high cholesterol content suppresses LDLR expression. Macrophage-derived foam cells are the most important cells and main cell type in atherosclerotic lesions. Uptake is initially beneficial as it sequesters damaging LDL but is harmful long-term as foam cells contribute to plaque mass. Furthermore, when foam cells die, they contribute to formation of necrotic material at plaque centre, which starts to destabilise atherosclerotic lesion. This forms a FATTY STREAK - a region of plaque comprising foam cells and lipid on the luminal surface that appears yellow. While these have little clinical effect as they do not protrude substantially into the lumen or impede blood flow and have a low chance of rupture, fatty streaks can progress into fibro-fatty plaques, which can lead to more serious clinical consequences.

Clinical consequences of plaque rupture - MI and stroke

More vulnerable plaques can rupture, forming complicated lesions. This happens when the ECM of the lesion breaks, usually at the shoulder of the fibrous cap that separates the lesion from the arterial lumen, exposing the highly thrombogenic contents of the plaque. This triggers rapid platelet adhesion and aggregation, forming an arterial thrombus (clot), which may occlude artery at site of plaque or become dislodged, forming an embolus that passes further down the arterial tree before occluding a vessel. Thrombi and emboli cause acute ischaemic events: Obstruction of coronary arteries supplying the heart muscle leads to myocardial infarction with ischaemic death of downstream myocardium. This impairs contractility and can lead to death. Obstruction of carotid arteries supplying the brain leads to ischaemic stroke.

Other cholesterol lowering drugs

Other cholesterol lowering drugs include bile acid sequestrants (e.g. colestipol). These are resins that bind bile constituents in the GI tract and prevent their reabsorption. This stimulates increased bile acid production by the liver, for which cholesterol is the substrate and so hepatic LDLR expression and LDL uptake is increased to meet demand. They are generally well tolerated but can cause GI problems, including constipation, diarrhoea and bloating. Anti PCSK9 antibodies are a novel lipid-lowering drug therapy. PCSK9 is an enzyme that binds to LDLRs and induces their degradation. Antibodies targeting PCSK9 (e.g. alirocumab) block this effect, which maintains higher LDLR membrane density, meaning more LDL is taken up by peripheral cells to reduce plasma LDL. Clinical trials showed promising efficacy (15% reduction in major adverse cv events, according to initial clinical trial) and they have been approved for use when other lipid-lowering therapies (statins or diet) are insufficient.

Historically used experimental models of atherosclerosis

Other, more historically used experimental models of atherosclerosis: Cholesterol-fed primates Cholesterol-fed or Watanabe (genetically mutant) rabbits Diabetic pigs on a high fat diet

Stability of plaques

SMCs help to stabilise plaques by producing a thick fibrous cap, which reduces tensile stress and prevents contact between the thrombogenic lipid-rich necrotic core of plaque and blood. This reduces the likelihood of secondary events, such as embolism or stroke. T cells and M1 macrophages, on the other hand, have destabilising action as they break down collagen and other ECM components in the shoulder region of plaques. This erodes the fibrous cap, generating a vulnerable lesion covered by a thin cap, which experiences tensile stress and is prone to rupture. Therefore, the ultimate plaque integrity is determined by the relative contribution of SMCs or macrophages/T cells (reparative or inflammatory processes).

Secondary treatment for atherosclerosis to limit complications

Secondary treatment is aimed at the complications of atherosclerosis.Several drugs can be used to prevent thrombosis and thus help to acutely treat some of the clinical consequences of ruptured atherosclerotic plaques. However, as they do not affect the plaque themselves, they cannot protect from other complications such as aneurysm. These include: anticoagulants (heparin, warfarin), anti-thrombotic drugs - anti platelet drugs (aspirin, abciximab) and thrombolytics (streptokinase, alteplase)

Statins

Statins (e.g. lovastatin, simvastatin) are lipid-lowering drugs that improve hypercholesterolaemia - prophylactic measure to reduce CGD development. They competitively inhibit hepatic HMG-CoA reductase, which catalyses the rate-limiting step in cholesterol synthesis, and so decrease cholesterol production and also promote hepatocyte LDLR expression. Together these effects decrease plasma LDL-cholesterol and TAG levels. They are usually well tolerated, but side effects include muscle problems, increased diabetes risk, and liver damage.

Surgical interventions for atherosclerosis

Surgical interventions can also be used to treat severe atherosclerotic disease. Balloon angioplasty is used to widen blocked or narrowed arteries (most commonly used in narrowed coronary arteries). A balloon is inserted into the narrowed vessel and when it inflates, the vessel wall is stretched outwards to expand lumen diameter. This may be combined with a stent to maintain vessel lumen size after widening. Coronary bypass is used to bypass a blocked coronary artery ( due to atherosclerotic lesion) via attachment of new conduit vessel (can be vein, or mammary/radial artery), which provides an alternative/collateral route for blood flow to perfuse tissue distal to the blockage.

Scandinavian Simvastatin Survival Study

The Scandinavian Simvastatin Survival Study (4S) confirmed the beneficial effects of statins in decreasing CHD and cardiovascular mortality. It found that secondary prevention (after first cv event) with simvastatin in a high risk group with CHD reduced overall mortality by 30%. Several other large multicentre clinical trials followed, leading to widespread use of simvastatin.

Faries et al. (2002) - VSMCs in atherosclerosis

The high proliferative and migratory capacity of VSMCs at atherosclerotic lesions was shown by Faries et al. (2002). Human VSMCs were isolated from atherosclerotic arteries and saphenous veins and compared for proliferation using total DNA fluorescence photometry and for migration using a Boyden chamber. Those from atherosclerotic arteries had greater proliferation and migration, although the comparison may not be entirely valid as the controls came from veins, which are not susceptible to atherosclerosis.

Mechanisms by which LDL cholesterol induces endothelial injury + EE for this

The mechanisms by which LDL cholesterol induces initial endothelial injury and dysfunction is not fully understood. It is known that hypercholesterolaemic stress converges with haemodynamic stress (from increased turbulence) to upregulate endothelial expression of adhesion molecules, which initiates atherogenesis. Increased LDL levels therefore promote plaque formation. Evidence for this comes from Fotis et al. (2012) who measured [ICAM1] and immunohistochemical expression of ICAM1 in the aortic endothelium of rats on four different diets. Group C with the highest cholesterol diet showed greatest ICAM-1 expression, while group D on the lowest cholesterol diet had the lowest expression. Although this study only shows association between dietary cholesterol content and ICAM1 expression, rather than actual incidence of atherosclerosis, when combined with other experimental data, it is evident that this action of LDL cholesterol combines with other factors to increase atherogenesis through adhesive factor expression. There may also be interactions between different types of stress to augment endothelial dysfunction.

Modifiable risk factors for atherosclerosis

There are several risk factors for atherosclerosis, which all promote endothelial dysfunction. Modifiable risk factors can be targeted by primary preventative strategies and so are of great public health interest. They include: Smoking - increases ROS and mLDL production and causes endothelial layer damage Obesity - augments hypertension and dyslipidaemia High cholesterol intake/hyperlipidaemia - especially with increase in LDL:HDL ratio (raised LDL and low HDL) and increased oxidised LDL levels. Hypertension - altered shear stress, which results in endothelial dysfunction and damage High alcohol intake Low physical activity - contributes to obesity Diabetes - increased risk of e

EE for endothelial activation

VCAM-1 is an example of a cell adhesion molecule and is a key marker of endothelial dysfunction. Damaged endothelium can therefore be identified by immunofluorescent staining using antibodies against VCAM-1. Staining WT mice for VCAM-1 shows that endothelial damage occurs at bifurcations and regions of low shear stress, but not in regions of high shear stress. This may be because high laminar shear stress activates anti-inflammatory and anti-atherogenic pathways, such as increasing vasodilatory NO production to prevent plaque formation.

Familial hypercholesterolemia

While it is common in the general population to have raised LDL, this can be exaggerated by mutations in genes encoding apoproteins and lipoproteins. Loss of function mutations in LDL receptors or ApoB100 (prevent LDL recognition at LDLR) or PCSK9 gain of function mutation (excessive lowering of LDLR expression) result in familial hypercholesterolemia. This increases risk of atherosclerotic plaque formation and the plethora of clinical consequences that can follow because LDL cannot be taken up by peripheral cells and so can become deposited in artery walls. Homozygotes for these mutations may experience acute vascular events as early as the first decade of life. These genetic defects also provide evidence for the role of hypercholesterolemia in atherogenesis and its progression.

Treg cells in atherosclerosis + EE

While most T effector cell responses aggravate atherosclerosis, Treg cells decrease inflammation and have anti-atherogenic effects - for example by promoting transformation of M1 to M2 macrophages. It is thought that their activity may be dysfunctional/downregulated in atherosclerosis. This is confirmed by evidence from mouse models of atherosclerosis, which show that Treg depletion exacerbates atherosclerosis and adoptive transfer of Tregs to ApoE -/- mice decreases atherosclerotic lesion size (Mallat et al. 2006). Replacing/restoring Treg numbers in this way could therefore stimulate plaque regression and reduce atherosclerosis. However, as with all animal models, there are limitations when applied to humans. Studies suggest that Tregs only have short survival durations after infusion as they may lose FOXP3 expression, which decreases their immunosuppressive ability and limits their anti-atherogenic role.